Peptidomimetic macrocycles

ABSTRACT

The present invention provides novel peptidomimetic macrocycles and methods of using such macrocycles for the treatment of disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/460,848 filed Aug. 15, 2014 which is a divisional of U.S. applicationSer. No. 13/816,880, filed Feb. 13, 2013 (or Apr. 25, 2013, which is the371 date), which is a national stage of PCT/US2011/047692, filed Aug.13, 2011, which claims the benefit of U.S. Provisional Application Nos.61/373,701, filed Aug. 13, 2010, 61/373,638, filed Aug. 13, 2010, and61/374,163, filed Aug. 16, 2010, each of which are hereby incorporatedby reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 31, 2013, isnamed 35224-757.831_SL.txt and is 586,330 bytes in size.

BACKGROUND OF THE INVENTION

The human transcription factor protein p53 induces cell cycle arrest andapoptosis in response to DNA damage and cellular stress, and therebyplays a critical role in protecting cells from malignant transformation.The E3 ubiquitin ligase HDM2 negatively regulates p53 function through adirect binding interaction that neutralizes the p53 transactivationactivity, leads to export from the nucleus of p53 protein, and targetsp53 for degradation via the ubiquitylation-proteasomal pathway. Loss ofp53 activity, either by deletion, mutation, or HDM2 overexpression, isthe most common defect in human cancers. Tumors that express wild typep53 are vulnerable to pharmacologic agents that stabilize or increasethe concentration of active p53. In this context, inhibition of theactivities of HDM2 has emerged as a validated approach to restore p53activity and resensitize cancer cells to apoptosis in vitro and in vivo.HDMX (HDM4) has more recently been identified as a similar negativeregulator of p53, and studies have revealed significant structuralhomology between the p53 binding interfaces of HDM2 and HDMX.

The p53-HDM2 and p53-HDMX protein-protein interactions are mediated bythe same 15-residue alpha-helical transactivation domain of p53, whichinserts into hydrophobic clefts on the surface of HDM2 and HDMX. Threeresidues within this domain of p53 (F19, W23, and L26) are essential forbinding to HDM2 and HDMX. The present invention provides p53-basedpeptidomimetic macrocycles that modulate the activities of p53 byinhibiting the interactions between p53 and HDM2, p53 and HDMX, or p53and both HDM2 and HDMX proteins, and that may be used for treatingdiseases including but not limited to cancer and otherhyperproliferative diseases.

SUMMARY OF THE INVENTION

Described below are stably cross-linked peptides related to a portion ofhuman p53 (“p53 peptidomimetic macrocycles”). These cross-linkedpeptides contain at least two modified amino acids that together form anintramolecular cross-link that can help to stabilize the alpha-helicalsecondary structure of a portion of p53 that is thought to be importantfor binding of p53 to HDM2 and for binding of p53 to HDMX. Accordingly,a cross-linked polypeptide described herein can have improved biologicalactivity relative to a corresponding polypeptide that is notcross-linked. The p53 peptidomimetic macrocycles are thought tointerfere with binding of p53 to HDM2 and/or of p53 to HDMX, therebyliberating functional p53 and inhibiting its destruction. The p53peptidomimetic macrocycles described herein can be used therapeutically,for example to treat cancers and other disorders characterized by anundesirably low level or a low activity of p53, and/or to treat cancersand other disorders characterized by an undesirably high level ofactivity of HDM2 or HDMX. The p53 peptidomimetic macrocycles may also beuseful for treatment of any disorder associated with disruptedregulation of the p53 transcriptional pathway, leading to conditions ofexcess cell survival and proliferation such as cancer and autoimmunity,in addition to conditions of inappropriate cell cycle arrest andapoptosis such as neurodegeneration and immunedeficiencies. In someinstances, the p53 peptidomimetic macrocycles bind to HDM2 (e.g.,GenBank® Accession No.: 228952; GI:228952) and/or HDMX (also referred toas HDM4; GenBank® Accession No.: 88702791; GI:88702791).

In one aspect, the present invention provides a peptidomimeticmacrocycle comprising an amino acid sequence which is at least about60%, 80%, 90%, or 95% identical to an amino acid sequence chosen fromthe group consisting of the amino acid sequences in Table 1, 2, 3, or 4.Alternatively, an amino acid sequence of said peptidomimetic macrocycleis chosen from the group consisting of the amino acid sequences inTable 1. Alternatively, an amino acid sequence of said peptidomimeticmacrocycle is chosen as above, and further wherein the macrocycle doesnot include a thioether or a triazole. In some embodiments, thepeptidomimetic macrocycle comprises a helix, such as an α-helix. Inother embodiments, the peptidomimetic macrocycle comprises anα,α-disubstituted amino acid. A peptidomimetic macrocycle of theinvention may comprise a crosslinker linking the α-positions of at leasttwo amino acids. At least one of said two amino acids may be anα,α-disubstituted amino acid.

In some embodiments, the peptidomimetic macrocycle has the formula:

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid, and the terminal D and E independently optionally include acapping group;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v and w are independently integers from 1-1000;

u is an integer from 1-10;

x, y and z are independently integers from 0-10; and

n is an integer from 1-5.

In various embodiments, the peptidomimetic macrocycle includes L₁ and L₂wherein L₁ and L₂ either alone or in combination do not include athioether or a triazole.

In other embodiments, the peptidomimetic macrocycle may comprise acrosslinker linking a backbone amino group of a first amino acid to asecond amino acid within the peptidomimetic macrocycle. For example, theinvention provides peptidomimetic macrocycles of the formula (IV) or(IVa):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid, and the terminal D and E independently optionally include acapping group;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

V and w are independently integers from 1-1000;

u is an integer from 1-10;

x, y and z are independently integers from 0-10; and

n is an integer from 1-5.

Additionally, the invention provides a method of treating cancer in asubject comprising administering to the subject a peptidomimeticmacrocycle of the invention. Also provided is a method of modulating theactivity of p53 or HDM2 or HDMX in a subject comprising administering tothe subject a peptidomimetic macrocycle of the invention, or a method ofantagonizing the interaction between p53 and HDM2 and/or HDMX proteinsin a subject comprising administering to the subject such apeptidomimetic macrocycle.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 describes the synthesis of Fmoc-Me-6-Chloro-Tryptophan &Fmoc-6-Chloro-Tryptophan.

FIG. 2 shows an LC-MS trace of Me-6-Chloro-(Boc)Tryptophan-Ni—S—BPB.

FIG. 3 shows a 1H-NMR spectrum of Me-6-Chloro-(Boc)Tryptophan-Ni—S—BPB.

FIG. 4 shows an LC-MS trace of Fmoc-Me-6-Chloro-(Boc)Tryptophan.

FIG. 5 shows a 1H-NMR spectrum of Fmoc-Me-6-Chloro-(Boc)Tryptophan.

FIGS. 6a-f describe the results of a cell viability assay, a competitionELISA assay, GRIP assay, Kd data, p21 activation assay, fluorescencepolarization competition binding and circular helicity data forexemplary peptidomimetic macrocycles of the invention (SEQ ID NOS38-178, respectively, in order of appearance).

FIGS. 7A-D provide data from a variety of macrocycles FIGS. 7A-7Bdisclose SEQ ID NOS 42, 163, 177, 214, 217, 344, 289-290, 383, 533, 529,543, 601, 544, 594, 279, 374 and 660, respectively in order ofappearance, and FIGS. 7C-7D disclose SEQ ID NOS 702, 699, 704, 706, 689,507, 624, 703, 716, 606, 605, 642, 691, 731, 375, 727, 662, 587 and 714,respectively in order of appearance).

FIGS. 8A-B provide data from a variety of macrocycles.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “macrocycle” refers to a molecule having achemical structure including a ring or cycle formed by at least 9covalently bonded atoms.

As used herein, the term “peptidomimetic macrocycle” or “crosslinkedpolypeptide” refers to a compound comprising a plurality of amino acidresidues joined by a plurality of peptide bonds and at least onemacrocycle-forming linker which forms a macrocycle between a firstnaturally-occurring or non-naturally-occurring amino acid residue (oranalog) and a second naturally-occurring or non-naturally-occurringamino acid residue (or analog) within the same molecule. Peptidomimeticmacrocycle include embodiments where the macrocycle-forming linkerconnects the α carbon of the first amino acid residue (or analog) to theα carbon of the second amino acid residue (or analog). Thepeptidomimetic macrocycles optionally include one or more non-peptidebonds between one or more amino acid residues and/or amino acid analogresidues, and optionally include one or more non-naturally-occurringamino acid residues or amino acid analog residues in addition to anywhich form the macrocycle. A “corresponding uncrosslinked polypeptide”when referred to in the context of a peptidomimetic macrocycle isunderstood to relate to a polypeptide of the same length as themacrocycle and comprising the equivalent natural amino acids of thewild-type sequence corresponding to the macrocycle.

As used herein, the term “stability” refers to the maintenance of adefined secondary structure in solution by a peptidomimetic macrocycleof the invention as measured by circular dichroism, NMR or anotherbiophysical measure, or resistance to proteolytic degradation in vitroor in vivo. Non-limiting examples of secondary structures contemplatedin this invention are α-helices, β-turns, and β-pleated sheets.

As used herein, the term “helical stability” refers to the maintenanceof α helical structure by a peptidomimetic macrocycle of the inventionas measured by circular dichroism or NMR. For example, in someembodiments, the peptidomimetic macrocycles of the invention exhibit atleast a 1.25, 1.5, 1.75 or 2-fold increase in α-helicity as determinedby circular dichroism compared to a corresponding uncrosslinkedmacrocycle.

The term “α-amino acid” or simply “amino acid” refers to a moleculecontaining both an amino group and a carboxyl group bound to a carbonwhich is designated the α-carbon. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the naturally-occurring aminoacids, as well as non-naturally occurring amino acids prepared byorganic synthesis or other metabolic routes. Unless the contextspecifically indicates otherwise, the term amino acid, as used herein,is intended to include amino acid analogs.

The term “naturally occurring amino acid” refers to any one of thetwenty amino acids commonly found in peptides synthesized in nature, andknown by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L,K, M, F, P, S, T, W, Y and V.

The term “amino acid analog” or “non-natural amino acid” refers to amolecule which is structurally similar to an amino acid and which can besubstituted for an amino acid in the formation of a peptidomimeticmacrocycle. Amino acid analogs include, without limitation, compoundswhich are structurally identical to an amino acid, as defined herein,except for the inclusion of one or more additional methylene groupsbetween the amino and carboxyl group (e.g., α-amino β-carboxy acids), orfor the substitution of the amino or carboxy group by a similarlyreactive group (e.g., substitution of the primary amine with a secondaryor tertiary amine, or substitution of the carboxy group with an ester).Non-natural amino acids include structures according to the following:

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide without abolishing orsubstantially altering its essential biological or biochemical activity(e.g., receptor binding or activation). An “essential” amino acidresidue is a residue that, when altered from the wild-type sequence ofthe polypeptide, results in abolishing or substantially abolishing thepolypeptide's essential biological or biochemical activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., K, R, H), acidic side chains (e.g., D, E), unchargedpolar side chains (e.g., G, N, Q, S, T, Y, C), nonpolar side chains(e.g., A, V, L, I, P, F, M, W), beta-branched side chains (e.g., T, V,I) and aromatic side chains (e.g., Y, F, W, H). Thus, a predictednonessential amino acid residue in a polypeptide, for example, ispreferably replaced with another amino acid residue from the same sidechain family. Other examples of acceptable substitutions aresubstitutions based on isosteric considerations (e.g. norleucine formethionine) or other properties (e.g. 2-thienylalanine forphenylalanine).

The term “capping group” refers to the chemical moiety occurring ateither the carboxy or amino terminus of the polypeptide chain of thesubject peptidomimetic macrocycle. The capping group of a carboxyterminus includes an unmodified carboxylic acid (ie —COOH) or acarboxylic acid with a substituent. For example, the carboxy terminusmay be substituted with an amino group to yield a carboxamide at theC-terminus. Various substituents include but are not limited to primaryand secondary amines, including pegylated secondary amines.Representative secondary amine capping groups for the C-terminusinclude:

The capping group of an amino terminus includes an unmodified amine (ie—NH₂) or an amine with a substituent. For example, the amino terminusmay be substituted with an acyl group to yield a carboxamide at theN-terminus. Various substituents include but are not limited tosubstituted acyl groups, including C₁-C₆ carbonyls, C₇-C₃₀ carbonyls,and pegylated carbamates. Representative capping groups for theN-terminus include:

The term “member” as used herein in conjunction with macrocycles ormacrocycle-forming linkers refers to the atoms that form or can form themacrocycle, and excludes substituent or side chain atoms. By analogy,cyclodecane, 1,2-difluoro-decane and 1,3-dimethyl cyclodecane are allconsidered ten-membered macrocycles as the hydrogen or fluorosubstituents or methyl side chains do not participate in forming themacrocycle.

The symbol “

” when used as part of a molecular structure refers to a single bond ora trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon in an amino acid. For example, the amino acid side chain foralanine is methyl, the amino acid side chain for phenylalanine isphenylmethyl, the amino acid side chain for cysteine is thiomethyl, theamino acid side chain for aspartate is carboxymethyl, the amino acidside chain for tyrosine is 4-hydroxyphenylmethyl, etc. Othernon-naturally occurring amino acid side chains are also included, forexample, those that occur in nature (e.g., an amino acid metabolite) orthose that are made synthetically (e.g., an α,□α di-substituted aminoacid).

The term “α,□α di-substituted amino” acid refers to a molecule or moietycontaining both an amino group and a carboxyl group bound to a carbon(the α-carbon) that is attached to two natural or non-natural amino acidside chains.

The term “polypeptide” encompasses two or more naturally ornon-naturally-occurring amino acids joined by a covalent bond (e.g., anamide bond). Polypeptides as described herein include full lengthproteins (e.g., fully processed proteins) as well as shorter amino acidsequences (e.g., fragments of naturally-occurring proteins or syntheticpolypeptide fragments).

The term “macrocyclization reagent” or “macrocycle-forming reagent” asused herein refers to any reagent which may be used to prepare apeptidomimetic macrocycle of the invention by mediating the reactionbetween two reactive groups. Reactive groups may be, for example, anazide and alkyne, in which case macrocyclization reagents include,without limitation, Cu reagents such as reagents which provide areactive Cu(I) species, such as CuBr, CuI or CuOTf, as well as Cu(II)salts such as Cu(CO₂CH₃)₂, CuSO₄, and CuCl₂ that can be converted insitu to an active Cu(I) reagent by the addition of a reducing agent suchas ascorbic acid or sodium ascorbate. Macrocyclization reagents mayadditionally include, for example, Ru reagents known in the art such asCp*RuCl(PPh₃)₂, [Cp*RuCl]₄ or other Ru reagents which may provide areactive Ru(II) species. In other cases, the reactive groups areterminal olefins. In such embodiments, the macrocyclization reagents ormacrocycle-forming reagents are metathesis catalysts including, but notlimited to, stabilized, late transition metal carbene complex catalystssuch as Group VIII transition metal carbene catalysts. For example, suchcatalysts are Ru and Os metal centers having a +2 oxidation state, anelectron count of 16 and pentacoordinated. Additional catalysts aredisclosed in Grubbs et al., “Ring Closing Metathesis and RelatedProcesses in Organic Synthesis” Acc. Chem. Res. 1995, 28, 446-452, andU.S. Pat. No. 5,811,515. In yet other cases, the reactive groups arethiol groups. In such embodiments, the macrocyclization reagent is, forexample, a linker functionalized with two thiol-reactive groups such ashalogen groups.

The term “halo” or “halogen” refers to fluorine, chlorine, bromine oriodine or a radical thereof.

The term “alkyl” refers to a hydrocarbon chain that is a straight chainor branched chain, containing the indicated number of carbon atoms. Forexample, C₁-C₁₀ indicates that the group has from 1 to 10 (inclusive)carbon atoms in it. In the absence of any numerical designation, “alkyl”is a chain (straight or branched) having 1 to 20 (inclusive) carbonatoms in it.

The term “alkylene” refers to a divalent alkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkenyl” refers to a C₂-C₆ alkenylchain. In the absence of any numerical designation, “alkenyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that is a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group has from 2 to 10 (inclusive)carbon atoms in it. The term “lower alkynyl” refers to a C₂-C₆ alkynylchain. In the absence of any numerical designation, “alkynyl” is a chain(straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring aresubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

“Arylalkyl” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with a C₁-C₅ alkylgroup, as defined above. Representative examples of an arylalkyl groupinclude, but are not limited to, 2-methylphenyl, 3-methylphenyl,4-methylphenyl, 2-ethylphenyl, 3-ethylphenyl, 4-ethylphenyl,2-propylphenyl, 3-propylphenyl, 4-propylphenyl, 2-butylphenyl,3-butylphenyl, 4-butylphenyl, 2-pentylphenyl, 3-pentylphenyl,4-pentylphenyl, 2-isopropylphenyl, 3-isopropylphenyl, 4-isopropylphenyl,2-isobutylphenyl, 3-isobutylphenyl, 4-isobutylphenyl, 2-sec-butylphenyl,3-sec-butylphenyl, 4-sec-butylphenyl, 2-t-butylphenyl, 3-t-butylphenyland 4-t-butylphenyl.

“Arylamido” refers to an aryl group, as defined above, wherein one ofthe aryl group's hydrogen atoms has been replaced with one or more—C(O)NH₂ groups. Representative examples of an arylamido group include2-C(O)NH2-phenyl, 3-C(O)NH₂-phenyl, 4-C(O)NH₂-phenyl, 2-C(O)NH₂-pyridyl,3-C(O)NH₂-pyridyl, and 4-C(O)NH₂-pyridyl,

“Alkylheterocycle” refers to a C₁-C₅ alkyl group, as defined above,wherein one of the C₁-C₅ alkyl group's hydrogen atoms has been replacedwith a heterocycle. Representative examples of an alkylheterocycle groupinclude, but are not limited to, —CH₂CH₂-morpholine, —CH₂CH₂-piperidine,—CH₂CH₂CH₂-morpholine, and —CH₂CH₂CH₂-imidazole.

“Alkylamido” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—C(O)NH₂ group. Representative examples of an alkylamido group include,but are not limited to, —CH₂—C(O)NH₂, —CH₂CH₂—C(O)NH₂,—CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂C(O)NH₂, —CH₂CH₂CH₂CH₂CH₂C(O)NH₂,—CH₂CH(C(O)NH₂)CH₃, —CH₂CH(C(O)NH₂)CH₂CH₃, —CH(C(O)NH₂)CH₂CH₃,—C(CH₃)₂CH₂C(O)NH₂, —CH₂—CH₂—NH—C(O)—CH₃, —CH₂—CH₂—NH—C(O)—CH₃—CH₃, and—CH₂—CH₂—NH—C(O)—CH═CH₂.

“Alkanol” refers to a C₁-C₅ alkyl group, as defined above, wherein oneof the C₁-C₅ alkyl group's hydrogen atoms has been replaced with ahydroxyl group. Representative examples of an alkanol group include, butare not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH(OH)CH₃, —CH₂CH(OH)CH₂CH₃, —CH(OH)CH₃ and—C(CH₃)₂CH₂OH.

“Alkylcarboxy” refers to a C₁-C₅ alkyl group, as defined above, whereinone of the C₁-C₅ alkyl group's hydrogen atoms has been replaced with a—COOH group. Representative examples of an alkylcarboxy group include,but are not limited to, —CH₂COOH, —CH₂CH₂COOH, —CH₂CH₂CH₂COOH,—CH₂CH₂CH₂CH₂COOH, —CH₂CH(COOH)CH₃, —CH₂CH₂CH₂CH₂CH₂COOH,—CH₂CH(COOH)CH₂CH₃, —CH(COOH)CH₂CH₃ and —C(CH₃)₂CH₂COOH.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally is optionally substituted. Somecycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, andcyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring are substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl.

The term “heteroarylalkoxy” refers to an alkoxy substituted withheteroaryl.

The term “heteroarylalkyl” or the term “heteroaralkyl” refers to analkyl substituted with a heteroaryl.

The term “heteroarylalkoxy” refers to an alkoxy substituted withheteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring are substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituent” refers to a group replacing a second atom orgroup such as a hydrogen atom on any molecule, compound or moiety.Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

In some embodiments, the compounds of this invention contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are included in thepresent invention unless expressly provided otherwise. In someembodiments, the compounds of this invention are also represented inmultiple tautomeric forms, in such instances, the invention includes alltautomeric forms of the compounds described herein (e.g., if alkylationof a ring system results in alkylation at multiple sites, the inventionincludes all such reaction products). All such isomeric forms of suchcompounds are included in the present invention unless expresslyprovided otherwise. All crystal forms of the compounds described hereinare included in the present invention unless expressly providedotherwise.

As used herein, the terms “increase” and “decrease” mean, respectively,to cause a statistically significantly (i.e., p<0.1) increase ordecrease of at least 5%.

As used herein, the recitation of a numerical range for a variable isintended to convey that the invention may be practiced with the variableequal to any of the values within that range. Thus, for a variable whichis inherently discrete, the variable is equal to any integer valuewithin the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, the variableis equal to any real value within the numerical range, including theend-points of the range. As an example, and without limitation, avariable which is described as having values between 0 and 2 takes thevalues 0, 1 or 2 if the variable is inherently discrete, and takes thevalues 0.0, 0.1, 0.01, 0.001, or any other real values ≥0 and ≤2 if thevariable is inherently continuous.

As used herein, unless specifically indicated otherwise, the word “or”is used in the inclusive sense of “and/or” and not the exclusive senseof “either/or.”

The term “on average” represents the mean value derived from performingat least three independent replicates for each data point.

The term “biological activity” encompasses structural and functionalproperties of a macrocycle of the invention. Biological activity is, forexample, structural stability, alpha-helicity, affinity for a target,resistance to proteolytic degradation, cell penetrability, intracellularstability, in vivo stability, or any combination thereof.

The details of one or more particular embodiments of the invention areset forth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

In some embodiments, the peptide sequences are derived from the p53protein.

A non-limiting exemplary list of suitable p53 peptides for use in thepresent invention is given below.

TABLE 1 (SEQ ID NOS 1-18, respectively, in order of appearance) Sequence(bold = critical residue; X = cross-linked amino acid) Design Notes Ac—Gln Ser Gln Gln Thr Phe Ser Asn Leu Trp Arg Leu Leu Pro Gln Asn —NH2linear Ac— X Gln Ser Gln X Thr Phe Ser Asn Leu Trp Arg Leu Leu Pro GlnAsn —NH2 i--> i + 4 x-link #1 Ac— X Ser Gln Gln X Phe Ser Asn Leu TrpArg Leu Leu Pro Gln Asn —NH2 i--> i + 4 x-link #2 Ac— Gln Ser X Gln ThrPhe X Asn Leu Trp Arg Leu Leu Pro Gln Asn —NH2 i--> i + 4 x-link #3 Ac—Gln Ser Gln X Thr Phe Ser X Leu Trp Arg Leu Leu Pro Gln Asn —NH2 i-->i + 4 x-link #4 Ac— Gln Ser Gln Gln X Phe Ser Asn X Trp Arg Leu Leu ProGln Asn —NH2 i--> i + 4 x-link #5 Ac— Gln Ser Gln Gln Thr Phe X Asn LeuTrp X Leu Leu Pro Gln Asn —NH2 i--> i + 4 x-link #6 Ac— Gln Ser Gln GlnThr Phe Ser X Leu Trp Arg X Leu Pro Gln Asn —NH2 i--> i + 4 x-link #7Ac— Gln Ser Gln Gln Thr Phe Ser Asn Leu Trp X Leu Leu Pro X Asn —NH2i--> i + 4 x-link #8 Ac— Gln Ser Gln Gln Thr Phe Ser Asn Leu Trp Arg XLeu Pro Gln X —NH2 i--> i + 4 x-link #9 Ac— X Gln Ser Gln Gln Thr Phe XAsn Leu Trp Arg Leu Leu Pro Gln Asn —NH2 i--> i + 7 x-link #1 Ac— X SerGln Gln Thr Phe Ser X Leu Trp Arg Leu Leu Pro Gln Asn —NH2 i--> i + 7x-link #2 Ac— Gln X Gln Gln Thr Phe Ser Asn X Trp Arg Leu Leu Pro GlnAsn —NH2 i--> i + 7 x-link #3 Ac— Gln Ser Gln X Thr Phe Ser Asn Leu TrpX Leu Leu Pro Gln Asn —NH2 i--> i + 7 x-link #4 Ac— Gln Ser Gln Gln XPhe Ser Asn Leu Trp Arg X Leu Pro Gln Asn —NH2 i--> i + 7 x-link #5 Ac—Gln Ser Gln Gln Thr Phe X Asn Leu Trp Arg Leu Leu X Gln Asn —NH2 i-->i + 7 x-link #6 Ac— Gln Ser Gln Gln Thr Phe Ser X Leu Trp Arg Leu LeuPro X Asn —NH2 i--> i + 7 x-link #7 Ac— Gln Ser Gln Gln Thr Phe Ser AsnX Trp Arg Leu Leu Pro Gln X —NH2 i--> i + 7 x-link #8

TABLE 2 (SEQ ID NOS 19-31, respectively, in order of appearance) DesignSequence (bold = critical residue; X = cross-linked amino acid) NotesAc— Leu Thr Phe Glu His Tyr Trp Ala Gln Leu Thr Ser —NH2 linear Ac— XLeu Thr Phe X His Tyr Trp Ala Gln Leu Thr Ser —NH2 i--> i + 4 x- link #1Ac— X Thr Phe Glu X Tyr Trp Ala Gln Leu Thr Ser —NH2 i--> i + 4 x- link#2 Ac— Leu X Phe Glu His X Trp Ala Gln Leu Thr Ser —NH2 i--> i + 4 x-link #3 Ac— Leu Thr Phe X His Tyr Trp X Gln Leu Thr Ser —NH2 i--> i + 4x- link #4 Ac— Leu Thr Phe Glu X Tyr Trp Ala X Leu Thr Ser —NH2 i--> i +4 x- link #5 Ac— Leu Thr Phe Glu His Tyr Trp X Gln Leu Thr X —NH2 i-->i + 4 x- link #6 Ac— Leu Thr Phe Glu His Tyr Trp Ala X Leu Thr Ser X—NH2 i--> i + 4 x- link #7 Ac— X Thr Phe Glu His Tyr Trp X Gln Leu ThrSer —NH2 i--> i + 7 x- link #1 Ac— Gln X Phe Glu His Tyr Trp Ala X LeuThr Ser —NH2 i--> i + 7 x- link #2 Ac— Gln Thr Phe X His Tyr Trp Ala GlnLeu X Ser —NH2 i--> i + 7 x- link #3 Ac— Gln Thr Phe Glu X Tyr Trp AlaGln Leu Thr X —NH2 i--> i + 7 x- link #4 Ac— Gln Thr Phe Glu His X TrpAla Gln Leu Thr Ser X —NH2 i--> i + 7 x- link #5

TABLE 3 (SEQ ID NOS 32-37, respectively, in order of appearance) DesignSequence (bold = critical residue; X = cross-linked amino acid) NotesAc— Phe Met Aib/His/ Tyr 6-ClTrp Glu Ac3c/Gln/Leu Leu —NH2 linear AsnAc— X Phe Met Aib/His/ X 6-ClTrp Glu Ac3c/Gln/Leu Leu —NH2 i--> i + 4 x-Asn link #1 Ac— Phe X Aib/His/ Tyr 6-ClTrp X Ac3c/Gln/Leu Leu —NH2 i-->i + 4 x- Asn link #2 Ac— Phe Met X Tyr 6-ClTrp Glu X Leu —NH2 i--> i + 4x- link #3 Ac— X Phe Met Aib/His/ Tyr 6-ClTrp Glu X Leu —NH2 i--> i + 7x- Asn link #1 Ac— Phe X Aib/His/ Tyr 6-ClTrp Glu Ac3c/Gln/Leu Leu X—NH2 i--> i + 7 x- Asn link #2

In Table 3 and elsewhere, “Aib” represents a 2-aminoisobutyric acidresidue, while “Ac3c” represents a aminocyclopropane carboxylic acidresidue.

Peptidomimetic Macrocycles

In some embodiments, a peptidomimetic macrocycle of the invention hasthe Formula (I):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid, and the terminal D and E independently optionally include acapping group;

B is a natural or non-natural amino acid, amino acid analog,

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with a Dresidue;

R₈ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅, or part of a cyclic structure with an Eresidue;

v and w are independently integers from 1-1000;

u is an integer from 1-10;

x, y and z are independently integers from 0-10; and

n is an integer from 1-5.

In one embodiment, L₁ and L₂, either alone or in combination, do notform a triazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 3. In otherembodiments of the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.Each occurrence of A, B, C, D or E in a macrocycle or macrocycleprecursor of the invention is independently selected. For example, asequence represented by the formula [A]_(x), when x is 3, encompassesembodiments where the amino acids are not identical, e.g. Gln-Asp-Ala aswell as embodiments where the amino acids are identical, e.g.Gln-Gln-Gln. This applies for any value of x, y, or z in the indicatedranges. Similarly, when u is greater than 1, each compound of theinvention may encompass peptidomimetic macrocycles which are the same ordifferent. For example, a compound of the invention may comprisepeptidomimetic macrocycles comprising different linker lengths orchemical compositions.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

In one embodiment, the peptidomimetic macrocycle of Formula (I) is:

wherein each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl,arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, orheterocycloalkyl, unsubstituted or substituted with halo-.

In related embodiments, the peptidomimetic macrocycle of Formula (I) is:

In other embodiments, the peptidomimetic macrocycle of Formula (I) is acompound of any of the formulas shown below:

wherein “AA” represents any natural or non-natural amino acid side chainand “

” is [D]_(v), [E]_(w) as defined above, and n is an integer between 0and 20, 50, 100, 200, 300, 400 or 500. In some embodiments, n is 0. Inother embodiments, n is less than 50.

Exemplary embodiments of the macrocycle-forming linker L are shownbelow.

In other embodiments, D and/or E in the compound of Formula I arefurther modified in order to facilitate cellular uptake. In someembodiments, lipidating or PEGylating a peptidomimetic macrocyclefacilitates cellular uptake, increases bioavailability, increases bloodcirculation, alters pharmacokinetics, decreases immunogenicity and/ordecreases the needed frequency of administration.

In other embodiments, at least one of [D] and [E] in the compound ofFormula I represents a moiety comprising an additionalmacrocycle-forming linker such that the peptidomimetic macrocyclecomprises at least two macrocycle-forming linkers. In a specificembodiment, a peptidomimetic macrocycle comprises two macrocycle-forminglinkers. In an embodiment, u is 2.

In the peptidomimetic macrocycles of the invention, any of themacrocycle-forming linkers described herein may be used in anycombination with any of the sequences shown in Tables 1-4 and also withany of the R— substituents indicated herein.

In some embodiments, the peptidomimetic macrocycle comprises at leastone α-helix motif. For example, A, B and/or C in the compound of FormulaI include one or more α-helices. As a general matter, α-helices includebetween 3 and 4 amino acid residues per turn. In some embodiments, theα-helix of the peptidomimetic macrocycle includes 1 to 5 turns and,therefore, 3 to 20 amino acid residues. In specific embodiments, theα-helix includes 1 turn, 2 turns, 3 turns, 4 turns, or 5 turns. In someembodiments, the macrocycle-forming linker stabilizes an α-helix motifincluded within the peptidomimetic macrocycle. Thus, in someembodiments, the length of the macrocycle-forming linker L from a firstCα to a second Cα is selected to increase the stability of an α-helix.In some embodiments, the macrocycle-forming linker spans from 1 turn to5 turns of the α-helix. In some embodiments, the macrocycle-forminglinker spans approximately 1 turn, 2 turns, 3 turns, 4 turns, or 5 turnsof the α-helix. In some embodiments, the length of themacrocycle-forming linker is approximately 5 Å to 9 Å per turn of theα-helix, or approximately 6 Å to 8 Å per turn of the α-helix. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelength is equal to approximately 5 carbon-carbon bonds to 13carbon-carbon bonds, approximately 7 carbon-carbon bonds to 11carbon-carbon bonds, or approximately 9 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 2 turns of an α-helix, thelength is equal to approximately 8 carbon-carbon bonds to 16carbon-carbon bonds, approximately 10 carbon-carbon bonds to 14carbon-carbon bonds, or approximately 12 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 3 turns of an α-helix, thelength is equal to approximately 14 carbon-carbon bonds to 22carbon-carbon bonds, approximately 16 carbon-carbon bonds to 20carbon-carbon bonds, or approximately 18 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 4 turns of an α-helix, thelength is equal to approximately 20 carbon-carbon bonds to 28carbon-carbon bonds, approximately 22 carbon-carbon bonds to 26carbon-carbon bonds, or approximately 24 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 5 turns of an α-helix, thelength is equal to approximately 26 carbon-carbon bonds to 34carbon-carbon bonds, approximately 28 carbon-carbon bonds to 32carbon-carbon bonds, or approximately 30 carbon-carbon bonds. Where themacrocycle-forming linker spans approximately 1 turn of an α-helix, thelinkage contains approximately 4 atoms to 12 atoms, approximately 6atoms to 10 atoms, or approximately 8 atoms. Where themacrocycle-forming linker spans approximately 2 turns of the α-helix,the linkage contains approximately 7 atoms to 15 atoms, approximately 9atoms to 13 atoms, or approximately 11 atoms. Where themacrocycle-forming linker spans approximately 3 turns of the α-helix,the linkage contains approximately 13 atoms to 21 atoms, approximately15 atoms to 19 atoms, or approximately 17 atoms. Where themacrocycle-forming linker spans approximately 4 turns of the α-helix,the linkage contains approximately 19 atoms to 27 atoms, approximately21 atoms to 25 atoms, or approximately 23 atoms. Where themacrocycle-forming linker spans approximately 5 turns of the α-helix,the linkage contains approximately 25 atoms to 33 atoms, approximately27 atoms to 31 atoms, or approximately 29 atoms. Where themacrocycle-forming linker spans approximately 1 turn of the α-helix, theresulting macrocycle forms a ring containing approximately 17 members to25 members, approximately 19 members to 23 members, or approximately 21members. Where the macrocycle-forming linker spans approximately 2 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 29 members to 37 members, approximately 31 members to 35members, or approximately 33 members. Where the macrocycle-forminglinker spans approximately 3 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 44 members to 52members, approximately 46 members to 50 members, or approximately 48members. Where the macrocycle-forming linker spans approximately 4 turnsof the α-helix, the resulting macrocycle forms a ring containingapproximately 59 members to 67 members, approximately 61 members to 65members, or approximately 63 members. Where the macrocycle-forminglinker spans approximately 5 turns of the α-helix, the resultingmacrocycle forms a ring containing approximately 74 members to 82members, approximately 76 members to 80 members, or approximately 78members.

In other embodiments, the invention provides peptidomimetic macrocyclesof Formula (IV) or (IVa):

wherein:

each A, C, D, and E is independently a natural or non-natural aminoacid, and the terminal D and E independently optionally include acapping group;

B is a natural or non-natural amino acid, amino acid analog.

[—NH-L₃-CO—], [—NH-L₃-SO₂—], or [—NH-L₃-];

R₁ and R₂ are independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-, or part of a cyclic structurewith an E residue;

R₃ is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, orheterocycloaryl, optionally substituted with R₅;

L is a macrocycle-forming linker of the formula -L₁-L₂-;

L₁ and L₂ are independently alkylene, alkenylene, alkynylene,heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene,heterocycloarylene, or [—R₄—K—R₄-]_(n), each being optionallysubstituted with R₅;

each R₄ is alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene;

each K is O, S, SO, SO₂, CO, CO₂, or CONR₃;

each R₅ is independently halogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆,—SO₂R₆, —CO₂R₆, a fluorescent moiety, a radioisotope or a therapeuticagent;

each R₆ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotopeor a therapeutic agent;

R₇ is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅;

v and w are independently integers from 1-1000;

u is an integer from 1-10;

x, y and z are independently integers from 0-10; and

n is an integer from 1-5.

In one example, L₁ and L₂, either alone or in combination, do not form atriazole or a thioether.

In one example, at least one of R₁ and R₂ is alkyl, unsubstituted orsubstituted with halo-. In another example, both R₁ and R₂ areindependently alkyl, unsubstituted or substituted with halo-. In someembodiments, at least one of R₁ and R₂ is methyl. In other embodiments,R₁ and R₂ are methyl.

In some embodiments of the invention, x+y+z is at least 1. In otherembodiments of the invention, x+y+z is at least 2. In other embodimentsof the invention, x+y+z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Eachoccurrence of A, B, C, D or E in a macrocycle or macrocycle precursor ofthe invention is independently selected. For example, a sequencerepresented by the formula [A]_(x), when x is 3, encompasses embodimentswhere the amino acids are not identical, e.g. Gln-Asp-Ala as well asembodiments where the amino acids are identical, e.g. Gln-Gln-Gln. Thisapplies for any value of x, y, or z in the indicated ranges.

In some embodiments, the peptidomimetic macrocycle of the inventioncomprises a secondary structure which is an α-helix and R₈ is —H,allowing intrahelical hydrogen bonding. In some embodiments, at leastone of A, B, C, D or E is an α,α-disubstituted amino acid. In oneexample, B is an α,α-disubstituted amino acid. For instance, at leastone of A, B, C, D or E is 2-aminoisobutyric acid. In other embodiments,at least one of A, B, C, D or E is

In other embodiments, the length of the macrocycle-forming linker L asmeasured from a first Cα to a second Cα is selected to stabilize adesired secondary peptide structure, such as an α-helix formed byresidues of the peptidomimetic macrocycle including, but not necessarilylimited to, those between the first Cα to a second Cα.

Exemplary embodiments of the macrocycle-forming linker -L₁-L₂- are shownbelow.

Preparation of Peptidomimetic Macrocycles

Peptidomimetic macrocycles of the invention may be prepared by any of avariety of methods known in the art. For example, any of the residuesindicated by “X” in Tables 1, 2, 3, or 4 may be substituted with aresidue capable of forming a crosslinker with a second residue in thesame molecule or a precursor of such a residue.

Various methods to effect formation of peptidomimetic macrocycles areknown in the art. For example, the preparation of peptidomimeticmacrocycles of Formula I is described in Schafmeister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdin, J. Am. Chem.Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004);U.S. Pat. No. 7,192,713 and PCT application WO 2008/121767. Theα,α-disubstituted amino acids and amino acid precursors disclosed in thecited references may be employed in synthesis of the peptidomimeticmacrocycle precursor polypeptides. For example, the “55-olefin aminoacid” is (S)-α-(2′-pentenyl) alanine and the “R8 olefin amino acid” is(R)-α-(2′-octenyl) alanine. Following incorporation of such amino acidsinto precursor polypeptides, the terminal olefins are reacted with ametathesis catalyst, leading to the formation of the peptidomimeticmacrocycle. In various embodiments, the following amino acids may beemployed in the synthesis of the peptidomimetic macrocycle:

In other embodiments, the peptidomimetic macrocyles of the invention areof Formula IV or IVa. Methods for the preparation of such macrocyclesare described, for example, in U.S. Pat. No. 7,202,332.

Additional methods of forming peptidomimetic macrocycles which areenvisioned as suitable to perform the present invention include thosedisclosed by Mustapa, M. Firouz Mohd et al., J. Org. Chem (2003), 68,pp. 8193-8198; Yang, Bin et al. Bioorg Med. Chem. Lett. (2004), 14, pp.1403-1406; U.S. Pat. No. 5,364,851; U.S. Pat. No. 5,446,128; U.S. Pat.No. 5,824,483; U.S. Pat. No. 6,713,280; and U.S. Pat. No. 7,202,332. Insuch embodiments, aminoacid precursors are used containing an additionalsubstituent R— at the alpha position. Such aminoacids are incorporatedinto the macrocycle precursor at the desired positions, which may be atthe positions where the crosslinker is substituted or, alternatively,elsewhere in the sequence of the macrocycle precursor. Cyclization ofthe precursor is then effected according to the indicated method.

Assays

The properties of the peptidomimetic macrocycles of the invention areassayed, for example, by using the methods described below. In someembodiments, a peptidomimetic macrocycle of the invention has improvedbiological properties relative to a corresponding polypeptide lackingthe substituents described herein.

Assay to Determine α-Helicity.

In solution, the secondary structure of polypeptides with α-helicaldomains will reach a dynamic equilibrium between random coil structuresand α-helical structures, often expressed as a “percent helicity”. Thus,for example, alpha-helical domains are predominantly random coils insolution, with α-helical content usually under 25%. Peptidomimeticmacrocycles with optimized linkers, on the other hand, possess, forexample, an alpha-helicity that is at least two-fold greater than thatof a corresponding uncrosslinked polypeptide. In some embodiments,macrocycles of the invention will possess an alpha-helicity of greaterthan 50%. To assay the helicity of peptidomimetic macrocyles of theinvention, the compounds are dissolved in an aqueous solution (e.g. 50mM potassium phosphate solution at pH 7, or distilled H₂O, toconcentrations of 25-50 μM). Circular dichroism (CD) spectra areobtained on a spectropolarimeter (e.g., Jasco J-710) using standardmeasurement parameters (e.g. temperature, 20° C.; wavelength, 190-260nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10;response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helicalcontent of each peptide is calculated by dividing the mean residueellipticity (e.g. [Φ]222 obs) by the reported value for a model helicaldecapeptide (Yang et al. (1986), Methods Enzymol. 130:208)).

Assay to Determine Melting Temperature (Tm).

A peptidomimetic macrocycle of the invention comprising a secondarystructure such as an α-helix exhibits, for example, a higher meltingtemperature than a corresponding uncrosslinked polypeptide. Typicallypeptidomimetic macrocycles of the invention exhibit Tm of >60° C.representing a highly stable structure in aqueous solutions. To assaythe effect of macrocycle formation on melting temperature,peptidomimetic macrocycles or unmodified peptides are dissolved indistilled H₂O (e.g. at a final concentration of 50 μM) and the Tm isdetermined by measuring the change in ellipticity over a temperaturerange (e.g. 4 to 95° C.) on a spectropolarimeter (e.g., Jasco J-710)using standard parameters (e.g. wavelength 222 nm; step resolution, 0.5nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1nm; temperature increase rate: 1° C./min; path length, 0.1 cm).

Protease Resistance Assay.

The amide bond of the peptide backbone is susceptible to hydrolysis byproteases, thereby rendering peptidic compounds vulnerable to rapiddegradation in vivo. Peptide helix formation, however, typically buriesthe amide backbone and therefore may shield it from proteolyticcleavage. The peptidomimetic macrocycles of the present invention may besubjected to in vitro trypsin proteolysis to assess for any change indegradation rate compared to a corresponding uncrosslinked polypeptide.For example, the peptidomimetic macrocycle and a correspondinguncrosslinked polypeptide are incubated with trypsin agarose and thereactions quenched at various time points by centrifugation andsubsequent HPLC injection to quantitate the residual substrate byultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocycleand peptidomimetic precursor (5 mcg) are incubated with trypsin agarose(Pierce) (S/E˜125) for 0, 10, 20, 90, and 180 minutes. Reactions arequenched by tabletop centrifugation at high speed; remaining substratein the isolated supernatant is quantified by HPLC-based peak detectionat 280 nm. The proteolytic reaction displays first order kinetics andthe rate constant, k, is determined from a plot of ln [S] versus time(k=−1×slope).

Ex Vivo Stability Assay.

Peptidomimetic macrocycles with optimized linkers possess, for example,an ex vivo half-life that is at least two-fold greater than that of acorresponding uncrosslinked polypeptide, and possess an ex vivohalf-life of 12 hours or more. For ex vivo serum stability studies, avariety of assays may be used. For example, a peptidomimetic macrocycleand a corresponding uncrosslinked polypeptide (2 mcg) are incubated withfresh mouse, rat and/or human serum (2 mL) at 37° C. for 0, 1, 2, 4, 8,and 24 hours. To determine the level of intact compound, the followingprocedure may be used: The samples are extracted by transferring 100 μlof sera to 2 ml centrifuge tubes followed by the addition of 10 μL of50% formic acid and 500 μL acetonitrile and centrifugation at 14,000 RPMfor 10 min at 4±2° C. The supernatants are then transferred to fresh 2ml tubes and evaporated on Turbovap under N₂<10 psi, 37° C. The samplesare reconstituted in 100 μL of 50:50 acetonitrile:water and submitted toLC-MS/MS analysis.

In Vitro Binding Assays.

To assess the binding and affinity of peptidomimetic macrocycles andpeptidomimetic precursors to acceptor proteins, a fluorescencepolarization assay (FPA) is used, for example. The FPA techniquemeasures the molecular orientation and mobility using polarized lightand fluorescent tracer. When excited with polarized light, fluorescenttracers (e.g., FITC) attached to molecules with high apparent molecularweights (e.g. FITC-labeled peptides bound to a large protein) emithigher levels of polarized fluorescence due to their slower rates ofrotation as compared to fluorescent tracers attached to smallermolecules (e.g. FITC-labeled peptides that are free in solution).

For example, fluoresceinated peptidomimetic macrocycles (25 nM) areincubated with the acceptor protein (25-1000 nM) in binding buffer (140mM NaCl, 50 mM Tris-HCL, pH 7.4) for 30 minutes at room temperature.Binding activity is measured, for example, by fluorescence polarizationon a luminescence spectrophotometer (e.g. Perkin-Elmer LS50B). Kd valuesmay be determined by nonlinear regression analysis using, for example,Graphpad Prism software (GraphPad Software, Inc., San Diego, Calif.). Apeptidomimetic macrocycle of the invention shows, in some instances,similar or lower Kd than a corresponding uncrosslinked polypeptide.

In Vitro Displacement Assays to Characterize Antagonists ofPeptide-Protein Interactions.

To assess the binding and affinity of compounds that antagonize theinteraction between a peptide and an acceptor protein, a fluorescencepolarization assay (FPA) utilizing a fluoresceinated peptidomimeticmacrocycle derived from a peptidomimetic precursor sequence is used, forexample. The FPA technique measures the molecular orientation andmobility using polarized light and fluorescent tracer. When excited withpolarized light, fluorescent tracers (e.g., FITC) attached to moleculeswith high apparent molecular weights (e.g. FITC-labeled peptides boundto a large protein) emit higher levels of polarized fluorescence due totheir slower rates of rotation as compared to fluorescent tracersattached to smaller molecules (e.g. FITC-labeled peptides that are freein solution). A compound that antagonizes the interaction between thefluoresceinated peptidomimetic macrocycle and an acceptor protein willbe detected in a competitive binding FPA experiment.

For example, putative antagonist compounds (1 nM to 1 mM) and afluoresceinated peptidomimetic macrocycle (25 nM) are incubated with theacceptor protein (50 nM) in binding buffer (140 mM NaCl, 50 mM Tris-HCL,pH 7.4) for 30 minutes at room temperature. Antagonist binding activityis measured, for example, by fluorescence polarization on a luminescencespectrophotometer (e.g. Perkin-Elmer LS50B). Kd values may be determinedby nonlinear regression analysis using, for example, Graphpad Prismsoftware (GraphPad Software, Inc., San Diego, Calif.).

Any class of molecule, such as small organic molecules, peptides,oligonucleotides or proteins can be examined as putative antagonists inthis assay.

Assay for Protein-Ligand Binding by Affinity Selection-Mass Spectrometry

To assess the binding and affinity of test compounds for proteins, anaffinity-selection mass spectrometry assay is used, for example.Protein-ligand binding experiments are conducted according to thefollowing representative procedure outlined for a system-wide controlexperiment using 1 μM peptidomimetic macrocycle plus 5 μM hMDM2. A 1 μLDMSO aliquot of a 40 μM stock solution of peptidomimetic macrocycle isdissolved in 19 μL of PBS (Phosphate-buffered saline: 50 mM, pH 7.5Phosphate buffer containing 150 mM NaCl). The resulting solution ismixed by repeated pipetting and clarified by centrifugation at 10 000 gfor 10 min. To a 4 μL aliquot of the resulting supernatant is added 4 μLof 10 μM hMDM2 in PBS. Each 8.0 μL experimental sample thus contains 40pmol (1.5 μg) of protein at 5.0 μM concentration in PBS plus 1 μMpeptidomimetic macrocycle and 2.5% DMSO. Duplicate samples thus preparedfor each concentration point are incubated for 60 mM at roomtemperature, and then chilled to 4° C. prior to size-exclusionchromatography-LC-MS analysis of 5.0 μL injections. Samples containing atarget protein, protein-ligand complexes, and unbound compounds areinjected onto an SEC column, where the complexes are separated fromnon-binding component by a rapid SEC step. The SEC column eluate ismonitored using UV detectors to confirm that the early-eluting proteinfraction, which elutes in the void volume of the SEC column, is wellresolved from unbound components that are retained on the column. Afterthe peak containing the protein and protein-ligand complexes elutes fromthe primary UV detector, it enters a sample loop where it is excisedfrom the flow stream of the SEC stage and transferred directly to theLC-MS via a valving mechanism. The (M+3H)³⁺ ion of the peptidomimeticmacrocycle is observed by ESI-MS at the expected m/z, confirming thedetection of the protein-ligand complex.

Assay for Protein-Ligand Kd Titration Experiments.

To assess the binding and affinity of test compounds for proteins, aprotein-ligand Kd titration experiment is performed, for example.Protein-ligand K_(d) titrations experiments are conducted as follows: 2μL DMSO aliquots of a serially diluted stock solution of titrantpeptidomimetic macrocycle (5, 2.5, . . . , 0.098 mM) are prepared thendissolved in 38 μL of PBS. The resulting solutions are mixed by repeatedpipetting and clarified by centrifugation at 10 000 g for 10 min. To 4.0μL aliquots of the resulting supernatants is added 4.0 μL of 10 μM hMDM2in PBS. Each 8.0 μL experimental sample thus contains 40 pmol (1.5 pig)of protein at 5.0 μM concentration in PBS, varying concentrations (125,62.5, . . . , 0.24 μM) of the titrant peptide, and 2.5% DMSO. Duplicatesamples thus prepared for each concentration point are incubated at roomtemperature for 30 min, then chilled to 4° C. prior to SEC-LC-MSanalysis of 2.0 μL injections. The (M+H)¹⁺, (M+2H)²⁺, (M+3H)³⁺, and/or(M+Na)¹⁺ ion is observed by ESI-MS; extracted ion chromatograms arequantified, then fit to equations to derive the binding affinity K_(d)as described in “A General Technique to Rank Protein-Ligand BindingAffinities and Determine Allosteric vs. Direct Binding Site Competitionin Compound Mixtures.” Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M.P.; Nash, H. M. J. Am. Chem. Soc. 2004, 126, 15495-15503; also in “ALIS:An Affinity Selection-Mass Spectrometry System for the Discovery andCharacterization of Protein-Ligand Interactions” D. A. Annis, C.-C.Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry.Edited by Wanner K, Höfner G: Wiley-VCH; 2007:121-184. Mannhold R,Kubinyi H, Folkers G (Series Editors): Methods and Principles inMedicinal Chemistry.

Assay for Competitive Binding Experiments by Affinity Selection-MassSpectrometry

To determine the ability of test compounds to bind competitively toproteins, an affinity selection mass spectrometry assay is performed,for example. A mixture of ligands at 40 μM per component is prepared bycombining 2 μL aliquots of 400 μM stocks of each of the three compoundswith 14 μL of DMSO. Then, 1 μL aliquots of this 40 μM per componentmixture are combined with 1 μL DMSO aliquots of a serially diluted stocksolution of titrant peptidomimetic macrocycle (10, 5, 2.5, . . . , 0.078mM). These 2 μL samples are dissolved in 38 μL of PBS. The resultingsolutions were mixed by repeated pipetting and clarified bycentrifugation at 10 000 g for 10 min. To 4.0 μL aliquots of theresulting supernatants is added 4.0 μL of 10 μM hMDM2 protein in PBS.Each 8.0 μL experimental sample thus contains 40 pmol (1.5 pig) ofprotein at 5.0 μM concentration in PBS plus 0.5 μM ligand, 2.5% DMSO,and varying concentrations (125, 62.5, . . . , 0.98 μM) of the titrantpeptidomimetic macrocycle. Duplicate samples thus prepared for eachconcentration point are incubated at room temperature for 60 min, thenchilled to 4° C. prior to SEC-LC-MS analysis of 2.0 μL injections.Additional details on these and other methods are provided in “A GeneralTechnique to Rank Protein-Ligand Binding Affinities and DetermineAllosteric vs. Direct Binding Site Competition in Compound Mixtures.”Annis, D. A.; Nazef, N.; Chuang, C. C.; Scott, M. P.; Nash, H. M. J. Am.Chem. Soc. 2004, 126, 15495-15503; also in “ALIS: An AffinitySelection-Mass Spectrometry System for the Discovery andCharacterization of Protein-Ligand Interactions” D. A. Annis, C.-C.Chuang, and N. Nazef. In Mass Spectrometry in Medicinal Chemistry.Edited by Wanner K, Höfner G: Wiley-VCH; 2007:121-184. Mannhold R,Kubinyi H, Folkers G (Series Editors): Methods and Principles inMedicinal Chemistry.

Binding Assays in Intact Cells.

It is possible to measure binding of peptides or peptidomimeticmacrocycles to their natural acceptors in intact cells byimmunoprecipitation experiments. For example, intact cells are incubatedwith fluoresceinated (FITC-labeled) compounds for 4 hrs in the absenceof serum, followed by serum replacement and further incubation thatranges from 4-18 hrs. Cells are then pelleted and incubated in lysisbuffer (50 mM Tris [pH 7.6], 150 mM NaCl, 1% CHAPS and proteaseinhibitor cocktail) for 10 minutes at 4° C. Extracts are centrifuged at14,000 rpm for 15 minutes and supernatants collected and incubated with10 μl goat anti-FITC antibody for 2 hrs, rotating at 4° C. followed byfurther 2 hrs incubation at 4° C. with protein A/G Sepharose (50 μl of50% bead slurry). After quick centrifugation, the pellets are washed inlysis buffer containing increasing salt concentration (e.g., 150, 300,500 mM). The beads are then re-equilibrated at 150 mM NaCl beforeaddition of SDS-containing sample buffer and boiling. Aftercentrifugation, the supernatants are optionally electrophoresed using4%-12% gradient Bis-Tris gels followed by transfer into Immobilon-Pmembranes. After blocking, blots are optionally incubated with anantibody that detects FITC and also with one or more antibodies thatdetect proteins that bind to the peptidomimetic macrocycle.

Cellular Penetrability Assays.

A peptidomimetic macrocycle is, for example, more cell penetrablecompared to a corresponding uncrosslinked macrocycle. Peptidomimeticmacrocycles with optimized linkers possess, for example, cellpenetrability that is at least two-fold greater than a correspondinguncrosslinked macrocycle, and often 20% or more of the appliedpeptidomimetic macrocycle will be observed to have penetrated the cellafter 4 hours. To measure the cell penetrability of peptidomimeticmacrocycles and corresponding uncrosslinked macrocycle, intact cells areincubated with fluoresceinated peptidomimetic macrocycles orcorresponding uncrosslinked macrocycle (10 μM) for 4 hrs in serum freemedia at 37° C., washed twice with media and incubated with trypsin(0.25%) for 10 mM at 37° C. The cells are washed again and resuspendedin PBS. Cellular fluorescence is analyzed, for example, by using eithera FACSCalibur flow cytometer or Cellomics' KineticScan HCS Reader.

Cellular Efficacy Assays.

The efficacy of certain peptidomimetic macrocycles is determined, forexample, in cell-based killing assays using a variety of tumorigenic andnon-tumorigenic cell lines and primary cells derived from human or mousecell populations. Cell viability is monitored, for example, over 24-96hrs of incubation with peptidomimetic macrocycles (0.5 to 50 μM) toidentify those that kill at EC50<10 μM. Several standard assays thatmeasure cell viability are commercially available and are optionallyused to assess the efficacy of the peptidomimetic macrocycles. Inaddition, assays that measure Annexin V and caspase activation areoptionally used to assess whether the peptidomimetic macrocycles killcells by activating the apoptotic machinery. For example, the CellTiter-glo assay is used which determines cell viability as a function ofintracellular ATP concentration.

In Vivo Stability Assay.

To investigate the in vivo stability of the peptidomimetic macrocycles,the compounds are, for example, administered to mice and/or rats by IV,IP, PO or inhalation routes at concentrations ranging from 0.1 to 50mg/kg and blood specimens withdrawn at 0′, 5′, 15′, 30′, 1 hr, 4 hrs, 8hrs and 24 hours post-injection. Levels of intact compound in 25 μL offresh serum are then measured by LC-MS/MS as above.

In Vivo Efficacy in Animal Models.

To determine the anti-oncogenic activity of peptidomimetic macrocyclesof the invention in vivo, the compounds are, for example, given alone(IP, IV, PO, by inhalation or nasal routes) or in combination withsub-optimal doses of relevant chemotherapy (e.g., cyclophosphamide,doxorubicin, etoposide). In one example, 5×10⁶ RS4; 11 cells(established from the bone marrow of a patient with acute lymphoblasticleukemia) that stably express luciferase are injected by tail vein inNOD-SCID mice 3 hrs after they have been subjected to total bodyirradiation. If left untreated, this form of leukemia is fatal in 3weeks in this model. The leukemia is readily monitored, for example, byinjecting the mice with D-luciferin (60 mg/kg) and imaging theanesthetized animals (e.g., Xenogen In Vivo Imaging System, Caliper LifeSciences, Hopkinton, Mass.). Total body bioluminescence is quantified byintegration of photonic flux (photons/sec) by Living Image Software(Caliper Life Sciences, Hopkinton, Mass.). Peptidomimetic macrocyclesalone or in combination with sub-optimal doses of relevantchemotherapeutics agents are, for example, administered to leukemic mice(10 days after injection/day 1 of experiment, in bioluminescence rangeof 14-16) by tail vein or IP routes at doses ranging from 0.1 mg/kg to50 mg/kg for 7 to 21 days. Optionally, the mice are imaged throughoutthe experiment every other day and survival monitored daily for theduration of the experiment. Expired mice are optionally subjected tonecropsy at the end of the experiment. Another animal model isimplantation into NOD-SCID mice of DoHH2, a cell line derived from humanfollicular lymphoma, that stably expresses luciferase. These in vivotests optionally generate preliminary pharmacokinetic, pharmacodynamicand toxicology data.

Clinical Trials.

To determine the suitability of the peptidomimetic macrocycles of theinvention for treatment of humans, clinical trials are performed. Forexample, patients diagnosed with cancer and in need of treatment areselected and separated in treatment and one or more control groups,wherein the treatment group is administered a peptidomimetic macrocycleof the invention, while the control groups receive a placebo or a knownanti-cancer drug. The treatment safety and efficacy of thepeptidomimetic macrocycles of the invention can thus be evaluated byperforming comparisons of the patient groups with respect to factorssuch as survival and quality-of-life. In this example, the patient grouptreated with a peptidomimetic macrocyle show improved long-term survivalcompared to a patient control group treated with a placebo.

Pharmaceutical Compositions and Routes of Administration

The peptidomimetic macrocycles of the invention also includepharmaceutically acceptable derivatives or prodrugs thereof. A“pharmaceutically acceptable derivative” means any pharmaceuticallyacceptable salt, ester, salt of an ester, pro-drug or other derivativeof a compound of this invention which, upon administration to arecipient, is capable of providing (directly or indirectly) a compoundof this invention. Particularly favored pharmaceutically acceptablederivatives are those that increase the bioavailability of the compoundsof the invention when administered to a mammal (e.g., by increasingabsorption into the blood of an orally administered compound) or whichincreases delivery of the active compound to a biological compartment(e.g., the brain or lymphatic system) relative to the parent species.Some pharmaceutically acceptable derivatives include a chemical groupwhich increases aqueous solubility or active transport across thegastrointestinal mucosa.

In some embodiments, the peptidomimetic macrocycles of the invention aremodified by covalently or non-covalently joining appropriate functionalgroups to enhance selective biological properties. Such modificationsinclude those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism, and alter rate ofexcretion.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, benzoate, benzenesulfonate, butyrate, citrate,digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate,heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, tosylate and undecanoate.Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄⁺ salts.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers include eithersolid or liquid carriers. Solid form preparations include powders,tablets, pills, capsules, cachets, suppositories, and dispersiblegranules. A solid carrier can be one or more substances, which also actsas diluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

Suitable solid excipients are carbohydrate or protein fillers include,but are not limited to sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethylcellulose; and gums including arabic and tragacanth;as well as proteins such as gelatin and collagen. If desired,disintegrating or solubilizing agents are added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

When the compositions of this invention comprise a combination of apeptidomimetic macrocycle and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 1 to 100%, and morepreferably between about 5 to 95% of the dosage normally administered ina monotherapy regimen. In some embodiments, the additional agents areadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents are part of asingle dosage form, mixed together with the compounds of this inventionin a single composition.

Methods of Use

In one aspect, the present invention provides novel peptidomimeticmacrocycles that are useful in competitive binding assays to identifyagents which bind to the natural ligand(s) of the proteins or peptidesupon which the peptidomimetic macrocycles are modeled. For example, inthe p53/HDMX system, labeled peptidomimetic macrocycles based on p53 canbe used in a HDMX binding assay along with small molecules thatcompetitively bind to HDMX. Competitive binding studies allow for rapidin vitro evaluation and determination of drug candidates specific forthe p53/HDMX system. Such binding studies may be performed with any ofthe peptidomimetic macrocycles disclosed herein and their bindingpartners.

The invention further provides for the generation of antibodies againstthe peptidomimetic macrocycles. In some embodiments, these antibodiesspecifically bind both the peptidomimetic macrocycle and the precursorpeptides, such as p53, to which the peptidomimetic macrocycles arerelated. Such antibodies, for example, disrupt the nativeprotein-protein interaction, for example, binding between p53 and HDMX.

In other aspects, the present invention provides for both prophylacticand therapeutic methods of treating a subject at risk of (or susceptibleto) a disorder or having a disorder associated with aberrant (e.g.,insufficient or excessive) expression or activity of the moleculesincluding p53, HDM2 or HDMX.

In another embodiment, a disorder is caused, at least in part, by anabnormal level of p53 or HDM2 or HDMX, (e.g., over or under expression),or by the presence of p53 or HDM2 or HDMX exhibiting abnormal activity.As such, the reduction in the level and/or activity of p53 or HDM2 orHDMX, or the enhancement of the level and/or activity of p53 or HDM2 orHDMX, by peptidomimetic macrocycles derived from p53, is used, forexample, to ameliorate or reduce the adverse symptoms of the disorder.

In another aspect, the present invention provides methods for treatingor preventing a disease including hyperproliferative disease andinflammatory disorder by interfering with the interaction or bindingbetween binding partners, for example, between p53 and HDM2 or p53 andHDMX. These methods comprise administering an effective amount of acompound of the invention to a warm blooded animal, including a human.In some embodiments, the administration of the compounds of the presentinvention induces cell growth arrest or apoptosis.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

In some embodiments, the peptidomimetics macrocycles of the invention isused to treat, prevent, and/or diagnose cancers and neoplasticconditions. As used herein, the terms “cancer”, “hyperproliferative” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. A metastatic tumor can arise from a multitude of primarytumor types, including but not limited to those of breast, lung, liver,colon and ovarian origin. “Pathologic hyperproliferative” cells occur indisease states characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair. Examples of cellular proliferative and/ordifferentiative disorders include cancer, e.g., carcinoma, sarcoma, ormetastatic disorders. In some embodiments, the peptidomimeticsmacrocycles are novel therapeutic agents for controlling breast cancer,ovarian cancer, colon cancer, lung cancer, metastasis of such cancersand the like.

Examples of cancers or neoplastic conditions include, but are notlimited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer,esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer,prostate cancer, uterine cancer, cancer of the head and neck, skincancer, brain cancer, squamous cell carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicularcancer, small cell lung carcinoma, non-small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma.

Examples of proliferative disorders include hematopoietic neoplasticdisorders. As used herein, the term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus (1991), Crit Rev.Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stembergdisease.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, noninflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps,adenomas, familial syndromes, colorectal carcinogenesis, colorectalcarcinoma, and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe ovary include, but are not limited to, ovarian tumors such as,tumors of coelomic epithelium, serous tumors, mucinous tumors,endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma,Brenner tumor, surface epithelial tumors; germ cell tumors such asmature (benign) teratomas, monodermal teratomas, immature malignantteratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sexcord-stomal tumors such as, granulosa-theca cell tumors,thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma;and metastatic tumors such as Krukenberg tumors.

In other or further embodiments, the peptidomimetics macrocyclesdescribed herein are used to treat, prevent or diagnose conditionscharacterized by overactive cell death or cellular death due tophysiologic insult, etc. Some examples of conditions characterized bypremature or unwanted cell death are or alternatively unwanted orexcessive cellular proliferation include, but are not limited tohypocellular/hypoplastic, acellular/aplastic, orhypercellular/hyperplastic conditions. Some examples include hematologicdisorders including but not limited to fanconi anemia, aplastic anemia,thalaessemia, congenital neutropenia, and myelodysplasia.

In other or further embodiments, the peptidomimetics macrocycles of theinvention that act to decrease apoptosis are used to treat disordersassociated with an undesirable level of cell death. Thus, in someembodiments, the anti-apoptotic peptidomimetics macrocycles of theinvention are used to treat disorders such as those that lead to celldeath associated with viral infection, e.g., infection associated withinfection with human immunodeficiency virus (HIV). A wide variety ofneurological diseases are characterized by the gradual loss of specificsets of neurons. One example is Alzheimer's disease (AD). Alzheimer'sdisease is characterized by loss of neurons and synapses in the cerebralcortex and certain subcortical regions. This loss results in grossatrophy of the affected regions. Both amyloid plaques andneurofibrillary tangles are visible in brains of those afflicted by ADAlzheimer's disease has been identified as a protein misfolding disease,due to the accumulation of abnormally folded A-beta and tau proteins inthe brain. Plaques are made up of β-amyloid. β-amyloid is a fragmentfrom a larger protein called amyloid precursor protein (APP). APP iscritical to neuron growth, survival and post-injury repair. In AD, anunknown process causes APP to be cleaved into smaller fragments byenzymes through proteolysis. One of these fragments is fibrils ofβ-amyloid, which form clumps that deposit outside neurons in denseformations known as senile plaques. Plaques continue to grow intoinsoluble twisted fibers within the nerve cell, often called tangles.Disruption of the interaction between β-amyloid and its native receptoris therefore important in the treatment of AD. The anti-apoptoticpeptidomimetics macrocycles of the invention are used, in someembodiments, in the treatment of AD and other neurological disordersassociated with cell apoptosis. Such neurological disorders includeAlzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis(ALS) retinitis pigmentosa, spinal muscular atrophy, and various formsof cerebellar degeneration. The cell loss in these diseases does notinduce an inflammatory response, and apoptosis appears to be themechanism of cell death.

In addition, a number of hematologic diseases are associated with adecreased production of blood cells. These disorders include anemiaassociated with chronic disease, aplastic anemia, chronic neutropenia,and the myelodysplastic syndromes. Disorders of blood cell production,such as myelodysplastic syndrome and some forms of aplastic anemia, areassociated with increased apoptotic cell death within the bone marrow.These disorders could result from the activation of genes that promoteapoptosis, acquired deficiencies in stromal cells or hematopoieticsurvival factors, or the direct effects of toxins and mediators ofimmune responses. Two common disorders associated with cell death aremyocardial infarctions and stroke. In both disorders, cells within thecentral area of ischemia, which is produced in the event of acute lossof blood flow, appear to die rapidly as a result of necrosis. However,outside the central ischemic zone, cells die over a more protracted timeperiod and morphologically appear to die by apoptosis. In other orfurther embodiments, the anti-apoptotic peptidomimetics macrocycles ofthe invention are used to treat all such disorders associated withundesirable cell death.

Some examples of neurologic disorders that are treated with thepeptidomimetics macrocycles described herein include but are not limitedto Alzheimer's Disease, Down's Syndrome, Dutch Type Hereditary CerebralHemorrhage Amyloidosis, Reactive Amyloidosis, Familial AmyloidNephropathy with Urticaria and Deafness, Muckle-Wells Syndrome,Idiopathic Myeloma; Macroglobulinemia-Associated Myeloma, FamilialAmyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, IsolatedCardiac Amyloid, Systemic Senile Amyloidosis, Adult Onset Diabetes,Insulinoma, Isolated Atrial Amyloid, Medullary Carcinoma of the Thyroid,Familial Amyloidosis, Hereditary Cerebral Hemorrhage With Amyloidosis,Familial Amyloidotic Polyneuropathy, Scrapie, Creutzfeldt-Jacob Disease,Gerstmann Straussler-Scheinker Syndrome, Bovine Spongiform Encephalitis,a prion-mediated disease, and Huntington's Disease.

In another embodiment, the peptidomimetics macrocycles described hereinare used to treat, prevent or diagnose inflammatory disorders. Numeroustypes of inflammatory disorders exist. Certain inflammatory diseases areassociated with the immune system, for example, autoimmune diseases.Autoimmune diseases arise from an overactive immune response of the bodyagainst substances and tissues normally present in the body, i.e. selfantigens. In other words, the immune system attacks its own cells.Autoimmune diseases are a major cause of immune-mediated diseases.Rheumatoid arthritis is an example of an autoimmune disease, in whichthe immune system attacks the joints, where it causes inflammation (i.e.arthritis) and destruction. It can also damage some organs, such as thelungs and skin. Rheumatoid arthritis can lead to substantial loss offunctioning and mobility. Rheumatoid arthritis is diagnosed with bloodtests especially the rheumatoid factor test. Some examples of autoimmunediseases that are treated with the peptidomimetics macrocycles describedherein include, but are not limited to, acute disseminatedencephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis,antiphospholipid antibody syndrome (APS), autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune inner ear disease, Bechet's disease,bullous pemphigoid, coeliac disease, Chagas disease, Churg-Strausssyndrome, chronic obstructive pulmonary disease (COPD), Crohn's disease,dermatomyositis, diabetes mellitus type 1, endometriosis, Goodpasture'ssyndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto'sdisease, Hidradenitis suppurativa, idiopathic thrombocytopenic purpura,inflammatory bowl disease (IBD), interstitial cystitis, lupuserythematosus, morphea, multiple sclerosis, myasthenia gravis,narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anaemia,Polymyositis, polymyalgia rheumatica, primary biliary cirrhosis,psoriasis, rheumatoid arthritis, schizophrenia, scleroderma, Sjögren'ssyndrome, temporal arteritis (also known as “giant cell arteritis”),Takayasu's arteritis, Vasculitis, Vitiligo, and Wegener'sgranulomatosis.

Some examples of other types of inflammatory disorders that are treatedwith the peptidomimetics macrocycles described herein include, but arenot limited to, allergy including allergic rhinitis/sinusitis, skinallergies (urticaria/hives, angioedema, atopic dermatitis), foodallergies, drug allergies, insect allergies, and rare allergic disorderssuch as mastocytosis, asthma, arthritis including osteoarthritis,rheumatoid arthritis, and spondyloarthropathies, primary angitis of theCNS, sarcoidosis, organ transplant rejection, fibromyalgia, fibrosis,pancreatitis, and pelvic inflammatory disease.

Examples of cardiovascular disorders (e.g., inflammatory disorders) thatare treated or prevented with the peptidomimetics macrocycles of theinvention include, but are not limited to, aortic valve stenosis,atherosclerosis, myocardial infarction, stroke, thrombosis, aneurism,heart failure, ischemic heart disease, angina pectoris, sudden cardiacdeath, hypertensive heart disease; non-coronary vessel disease, such asarteriolosclerosis, small vessel disease, nephropathy,hypertriglyceridemia, hypercholesterolemia, hyperlipidemia,xanthomatosis, asthma, hypertension, emphysema and chronic pulmonarydisease; or a cardiovascular condition associated with interventionalprocedures (“procedural vascular trauma”), such as restenosis followingangioplasty, placement of a shunt, stent, synthetic or natural excisiongrafts, indwelling catheter, valve or other implantable devices.Preferred cardiovascular disorders include atherosclerosis, myocardialinfarction, aneurism, and stroke.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1: Synthesis of 6-Chlorotryptophan Fmoc Amino Acids

Tert-butyl 6-chloro-3-formyl-1H-indole-1-carboxylate, 1. To a stirredsolution of dry DMF (12 mL) was added dropwise POCl₃ (3.92 mL, 43 mmol,1.3 equiv) at 0° C. under Argon. The solution was stirred at the sametemperature for 20 min before a solution of 6-chloroindole (5.0 g, 33mmol, 1 eq.) in dry DMF (30 mL) was added dropwise. The resultingmixture was allowed to warm to room temperature and stirred for anadditional 2.5 h. Water (50 mL) was added and the solution wasneutralized with 4M aqueous NaOH (pH˜8). The resulting solid wasfiltered off, washed with water and dried under vacuum. This materialwas directly used in the next step without additional purification. To astirred solution of the crude formyl indole (33 mmol, 1 eq.) in THF (150mL) was added successively Boc₂O (7.91 g, 36.3 mmol, 1.1 equiv) and DMAP(0.4 g, 3.3 mmol, 0.1 equiv) at room temperature under N₂. The resultingmixture was stirred at room temperature for 1.5 h and the solvent wasevaporated under reduced pressure. The residue was taken up in EtOAc andwashed with 1N HCl, dried and concentrated to give the formyl indole 1(9 g, 98% over 2 steps) as a white solid. ¹H NMR (CDCl₃) δ: 1.70 (s,Boc, 9H); 7.35 (dd, 1H); 8.21 (m, 3H); 10.07 (s, 1H).

Tert-butyl 6-chloro-3-(hydroxymethyl)-1H-indole-1-carboxylate, 2. To asolution of compound 1 (8.86 g, 32 mmol, 1 eq.) in ethanol (150 mL) wasadded NaBH₄ (2.4 g, 63 mmol, 2 eq.). The reaction was stirred for 3 h atroom temperature. The reaction mixture was concentrated and the residuewas poured into diethyl ether and water. The organic layer wasseparated, dried over magnesium sulfate and concentrated to give a whitesolid (8.7 g, 98%). This material was directly used in the next stepwithout additional purification. ¹H NMR (CDCl₃) δ: 1.65 (s, Boc, 9H);4.80 (s, 2H, CH₂); 7.21 (dd, 1H); 7.53 (m, 2H); 8.16 (bs, 1H).

Tert-butyl 3-(bromomethyl)-6-chloro-1H-indole-1-carboxylate, 3. To asolution of compound 2 (4.1 g, 14.6 mmol, 1 eq.) in dichloromethane (50mL) under argon was added a solution of triphenylphosphine (4.59 g, 17.5mmol, 1.2 eq.) in dichloromethane (50 mL) at −40° C. The reactionsolution was stirred an additional 30 min at 40° C. Then NBS (3.38 g, 19mmol, 1.3 eq.) was added. The resulting mixture was allowed to warm toroom temperature and stirred overnight. Dichloromethane was evaporated,Carbon Tetrachloride (100 mL) was added and the mixture was stirred for1 h and filtrated. The filtrate was concentrated, loaded in a silicaplug and quickly eluted with 25% EtOAc in Hexanes. The solution wasconcentrated to give a white foam (3.84 g, 77%). ¹H NMR (CDCl₃) δ: 1.66(s, Boc, 9H); 4.63 (s, 2H, CH₂); 7.28 (dd, 1H); 7.57 (d, 1H); 7.64 (bs,1H); 8.18 (bs, 1H).

αMe-6Cl-Trp(Boc)-Ni—S—BPB, 4. To S-Ala-Ni—S—BPB (2.66 g, 5.2 mmol, 1eq.) and KO-tBu (0.87 g, 7.8 mmol, 1.5 eq.) was added 50 mL of DMF underargon. The bromide derivative compound 3 (2.68 g, 7.8 mmol, 1.5 eq.) insolution of DMF (5.0 mL) was added via syringe. The reaction mixture wasstirred at ambient temperature for 1 h. The solution was then quenchedwith 5% aqueous acetic acid and diluted with water. The desired productwas extracted in dichloromethane, dried and concentrated. The oilyproduct 4 was purified by flash chromatography (solid loading) on normalphase using EtOAc and Hexanes as eluents to give a red solid (1.78 g,45% yield). αMe-6Cl-Trp(Boc)-Ni—S—BPB, 4: M+H calc. 775.21, M+H obs.775.26; ¹H NMR (CDCl₃) δ: 1.23 (s, 3H, αMe); 1.56 (m, 11H, Boc+CH₂);1.82-2.20 (m, 4H, 2CH₂); 3.03 (m, 1H, CH_(α)); 3.24 (m, 2H, CH₂); 3.57and 4.29 (AB system, 2H, CH₂ (benzyl), J=12.8 Hz); 6.62 (d, 2H); 6.98(d, 1H); 7.14 (m, 2H); 7.23 (m, 1H); 7.32-7.36 (m, 5H); 7.50 (m, 2H);7.67 (bs, 1H); 7.98 (d, 2H); 8.27 (m, 2H).

Fmoc-αMe-6Cl-Trp(Boc)-OH, 6. To a solution of 3N HCl/MeOH (1/3, 15 mL)at 50° C. was added a solution of compound 4 (1.75 g, 2.3 mmol, 1 eq.)in MeOH (5 ml) dropwise. The starting material disappeared within 3-4 h.The acidic solution was then cooled to 0° C. with an ice bath andquenched with an aqueous solution of Na₂CO₃ (1.21 g, 11.5 mmol, 5 eq.).Methanol was removed and 8 more equivalents of Na₂CO₃ (1.95 g, 18.4mmol) were added to the suspension. The Nickel scavenging EDTA disodiumsalt dihydrate (1.68 g, 4.5 mmol, 2 eq.) was then added and thesuspension was stirred for 2 h. A solution of Fmoc-OSu (0.84 g, 2.5mmol, 1.1 eq.) in acetone (50 mL) was added and the reaction was stirredovernight. Afterwards, the reaction was diluted with diethyl ether and1N HCl. The organic layer was then dried over magnesium sulfate andconcentrated in vacuo. The desired product 6 was purified on normalphase using acetone and dichloromethane as eluents to give a white foam(0.9 g, 70% yield). Fmoc-αMe-6Cl-Trp(Boc)-OH, 6: M+H calc. 575.19, M+Hobs. 575.37; ¹H NMR (CDCl₃) 1.59 (s, 9H, Boc); 1.68 (s, 3H, Me); 3.48(bs, 2H, CH₂); 4.22 (m, 1H, CH); 4.39 (bs, 2H, CH₂); 5.47 (s, 1H, NH);7.10 (m, 1H); 7.18 (m, 2H); 7.27 (m, 2H); 7.39 (m, 2H); 7.50 (m, 2H);7.75 (d, 2H); 8.12 (bs, 1H).

6Cl-Trp(Boc)-Ni—S—BPB, 5. To Gly-Ni—S—BPB (4.6 g, 9.2 mmol, 1 eq.) andKO-tBu (1.14 g, 10.1 mmol, 1.1 eq.) was added 95 mL of DMF under argon.The bromide derivative compound 3 (3.5 g, 4.6 mmol, 1.1 eq.) in solutionof DMF (10 mL) was added via syringe. The reaction mixture was stirredat ambient temperature for 1 h. The solution was then quenched with 5%aqueous acetic acid and diluted with water. The desired product wasextracted in dichloromethane, dried and concentrated. The oily product 5was purified by flash chromatography (solid loading) on normal phaseusing EtOAc and Hexanes as eluents to give a red solid (5 g, 71% yield).6Cl-Trp(Boc)-Ni—S—BPB, 5: M+H calc. 761.20, M+H obs. 761.34; ¹H NMR(CDCl₃) δ: 1.58 (m, 11H, Boc+CH₂); 1.84 (m, 1H); 1.96 (m, 1H); 2.24 (m,2H, CH₂); 3.00 (m, 1H, CH_(α)); 3.22 (m, 2H, CH₂); 3.45 and 4.25 (ABsystem, 2H, CH₂ (benzyl), J=12.8 Hz); 4.27 (m, 1H, CH_(α)); 6.65 (d,2H); 6.88 (d, 1H); 7.07 (m, 2H); 7.14 (m, 2H); 7.28 (m, 3H); 7.35-7.39(m, 2H); 7.52 (m, 2H); 7.96 (d, 2H); 8.28 (m, 2H).

Fmoc-6Cl-Trp(Boc)-OH, 7. To a solution of 3N HCl/MeOH (1/3, 44 mL) at50° C. was added a solution of compound 5 (5 g, 6.6 mmol, 1 eq.) in MeOH(10 ml) dropwise. The starting material disappeared within 3-4 h. Theacidic solution was then cooled to 0° C. with an ice bath and quenchedwith an aqueous solution of Na₂CO₃ (3.48 g, 33 mmol, 5 eq.). Methanolwas removed and 8 more equivalents of Na₂CO₃ (5.57 g, 52 mmol) wereadded to the suspension. The Nickel scavenging EDTA disodium saltdihydrate (4.89 g, 13.1 mmol, 2 eq.) and the suspension was stirred for2 h. A solution of Fmoc-OSu (2.21 g, 6.55 mmol, 1.1 eq.) in acetone (100mL) was added and the reaction was stirred overnight. Afterwards, thereaction was diluted with diethyl ether and 1N HCl. The organic layerwas then dried over magnesium sulfate and concentrated in vacuo. Thedesired product 7 was purified on normal phase using acetone anddichloromethane as eluents to give a white foam (2.6 g, 69% yield).Fmoc-6Cl-Trp(Boc)-OH, 7: M+H calc. 561.17, M+H obs. 561.37; ¹H NMR(CDCl₃) 1.63 (s, 9H, Boc); 3.26 (m, 2H, CH₂); 4.19 (m, 1H, CH); 4.39 (m,2H, CH₂); 4.76 (m, 1H); 5.35 (d, 1H, NH); 7.18 (m, 2H); 7.28 (m, 2H);7.39 (m, 3H); 7.50 (m, 2H); 7.75 (d, 2H); 8.14 (bs, 1H).

Example 2: Peptidomimetic Macrocycles of the Invention

Peptidomimetic macrocycles were synthesized, purified and analyzed aspreviously described and as described below (Schafmeister et al., J. Am.Chem. Soc. 122:5891-5892 (2000); Schafmeister & Verdine, J. Am. Chem.Soc. 122:5891 (2005); Walensky et al., Science 305:1466-1470 (2004); andU.S. Pat. No. 7,192,713). Peptidomimetic macrocycles were designed byreplacing two or more naturally occurring amino acids with thecorresponding synthetic amino acids. Substitutions were made at i andi+4, and i and i+7 positions. Peptide synthesis was performed eithermanually or on an automated peptide synthesizer (Applied Biosystems,model 433A), using solid phase conditions, rink amide AM resin(Novabiochem), and Fmoc main-chain protecting group chemistry. For thecoupling of natural Fmoc-protected amino acids (Novabiochem), 10equivalents of amino acid and a 1:1:2 molar ratio of coupling reagentsHBTU/HOBt (Novabiochem)/DIEA were employed. Non-natural amino acids (4equiv) were coupled with a 1:1:2 molar ratio of HATU (AppliedBiosystems)/HOBt/DIEA. The N-termini of the synthetic peptides wereacetylated, while the C-termini were amidated.

Purification of cross-linked compounds was achieved by high performanceliquid chromatography (HPLC) (Varian ProStar) on a reverse phase C18column (Varian) to yield the pure compounds. Chemical composition of thepure products was confirmed by LC/MS mass spectrometry (Micromass LCTinterfaced with Agilent 1100 HPLC system) and amino acid analysis(Applied Biosystems, model 420A).

Table 4 shows a list of peptidomimetic macrocycles of the inventionprepared.

TABLE 4 SEQ ID Exact Qbserved NQ: SP Seq Mass M + 2 mass (m/e)  38 SP-1Ac-LSQETF$r8DLWKLL$EN-NH2 2068.13 1035.07 1035.36  39 SP-2Ac-LSQETF$r8NLWKLL$QN-NH2 2066.16 1034.08 1034.31  40 SP-3Ac-LSQQTF$r8NLWRLL$QN-NH2 2093.18 1047.59 1047.73  41 SP-4Ac-QSQQTF$r8NLWKLL$QN-NH2 2080.15 1041.08 1041.31  42 SP-5Ac-QSQQTF$r8NLWRLLSQN-NH2 2108.15 1055.08 1055.32  43 SP-6Ac-QSQQTA$r8NLWRLL$QN-NH2 2032.12 1017.06 1017.24  44 SP-7Ac-QAibQQTF$r8NLWRLL$QN-NH2 2106.17 1054.09 1054.34  45 SP-8Ac-QSQQTFSNLWRLLPQN-NH2 2000.02 1001.01 1001.26  46 SP-9Ac-QSQQTF$/r8NLWRLLVQN-NH2 2136.18 1069.09 1069.37  47 SP-10Ac-QSQAibTF$r8NLWRLL$QN-NH2 2065.15 1033.58 1033.71  48 SP-11Ac-QSQQTF$r8NLWRLL$AN-NH2 2051.13 1026.57 1026.70  49 SP-12Ac-ASQQTF$r8NLWRLL$QN-NH2 2051.13 1026.57 1026.90  50 SP-13Ac-QSQQTF$r8ALWRLL$QN-NH2 2065.15 1033.58 1033.41  51 SP-14Ac-QSQETF$r8NLWRLL$QN-NH2 2109.14 1055.57 1055.70  52 SP-15Ac-RSQQTF$r8NLWRLL$QN-NH2 2136.20 1069.10 1069.17  53 SP-16Ac-RSQQTF$r8NLWRLL$EN-NH2 2137.18 1069.59 1069.75  54 SP-17Ac-LSQETFSDLWKLLPEN-NH2 1959.99 981.00 981.24  55 SP-18Ac-QSQ$TFS$LWRLLPQN-NH2 2008.09 1005.05 1004.97  56 SP-19Ac-QSQQ$FSN$WRLLPQN-NH2 2036.06 1019.03 1018.86  57 SP-20Ac-QSQQT$SNL$RLLPQN-NH2 1917.04 959.52 959.32  58 SP-21Ac-QSQQTF$NLW$LLPQN-NH2 2007.06 1004.53 1004.97  59 SP-22Ac-RTQATF$r8NQWAibANle$TNAibTR- NH2 2310.26 1156.13 1156.52  60 SP-23Ac-QSQQTF$r8NLWRLL$RN-NH2 2136.20 1069.10 1068.94  61 SP-24Ac-QSQRTF$r8NLWRLL$QN-NH2 2136.20 1069.10 1068.94  62 SP-25Ac-QSQQTF$r8NNleWRLL$QN-NH2 2108.15 1055.08 1055.44  63 SP-26Ac-QSQQTF$r8NLWRNleL$QN-NH2 2108.15 1055.08 1055.84  64 SP-27Ac-QSQQTF$r8NLWRLNle$QN-NH2 2108.15 1055.08 1055.12  65 SP-28Ac-QSQQTY$r8NLWRLL$QN-NH2 2124.15 1063.08 1062.92  66 SP-29Ac-RAibQQTF$r8NLWRLL$QN-NH2 2134.22 1068.11 1068.65  67 SP-30Ac-MPRFMDYWEGLN-NH2 1598.70 800.35 800.45  68 SP-31Ac-RSQQRF$r8NLWRLL$QN-NH2 2191.25 1096.63 1096.83  69 SP-32Ac-QSQQRF$r8NLWRLL$QN-NH2 2163.21 1082.61 1082.87  70 SP-33Ac-RAibQQRF$r8NLWRLL$QN-NH2 2189.27 1095.64 1096.37  71 SP-34Ac-RSQQRF$r8NFWRLL$QN-NH2 2225.23 1113.62 1114.37  72 SP-35Ac-RSQQRF$r8NYWRLL$QN-NH2 2241.23 1121.62 1122.37  73 SP-36Ac-RSQQTF$r8NLWQLL$QN-NH2 2108.15 1055.08 1055.29  74 SP-37Ac-QSQQTF$r8NLWQAmlL$QN-NH2 2094.13 1048.07 1048.32  75 SP-38Ac-QSQQTF$r8NAmlWRLL$QN-NH2 2122.17 1062.09 1062.35  76 SP-39Ac-NlePRF$r8DYWEGL$QN-NH2 1869.98 935.99 936.20  77 SP-40Ac-NlePRF$r8NYWRLL$QN-NH2 1952.12 977.06 977.35  78 SP-41Ac-RF$r8NLWRLL$Q-NH2 1577.96 789.98 790.18  79 SP-42Ac-QSQQTF$r8N2ffWRLL$QN-NH2 2160.13 1081.07 1081.40  80 SP-43Ac-QSQQTF$r8N3ffWRLL$QN-NH2 2160.13 1081.07 1081.34  81 SP-44Ac-QSQQTF#r8NLWRLL#QN-NH2 2080.12 1041.06 1041.34  82 SP-45Ac-RSQQTA$r8NLWRLL$QN-NH2 2060.16 1031.08 1031.38  83 SP-46Ac-QSQQTF%r8NLWRLL%QN-NH2 2110.17 1056.09 1056.55  84 SP-47HepQSQ$TFSNLWRLLPQN-NH2 2051.10 1026.55 1026.82  85 SP-48HepQSQ$TF$r8NLWRLL$QN-NH2 2159.23 1080.62 1080.89  86 SP-49Ac-QSQQTF$r8NL6clWRLL$QN-NH2 2142.11 1072.06 1072.35  87 SP-50Ac-QSQQTF$r8NLMe6clwRLL$QN-NH2 2156.13 1079.07 1079.27  88 SP-51Ac-LTFEHYWAQLTS-NH2 1535.74 768.87 768.91  89 SP-52 Ac-LTF$HYW$QLTS-NH21585.83 793.92 794.17  90 SP-53 Ac-LTFE$YWA$LTS-NH2 1520.79 761.40761.67  91 SP-54 Ac-LTF$zr8HYWAQL$zS-NH2 1597.87 799.94 800.06  92 SP-55Ac-LTF$r8HYWRQL$S-NH2 1682.93 842.47 842.72  93 SP-56Ac-QS$QTFStNLWRLL$s8QN-NH2 2145.21 1073.61 1073.90  94 SP-57Ac-QSQQTASNLWRLLPQN-NH2 1923.99 963.00 963.26  95 SP-58Ac-QSQQTA$/r8NLWRLL$/QN-NH2 2060.15 1031.08 1031.24  96 SP-59Ac-ASQQTFS/r8NLWRLLS/QN-NH2 2079.16 1040.58 1040.89  97 SP-60Ac-SSQQSFSNLWRLLAibQN-NH2 2009.09 1005.55 1005.86  98 SP-61Ac-QSSQTFSNLWRLLAihQN-NH2 2023.10 1012.55 1012.79  99 SP-62Ac-QSQQ$FSNSWRLLAibQN-NH2 2024.06 1013.03 1013.31 100 SP-63Ac-QSQQTFSNLWSLLAibQN-NH2 1995.06 998.53 998.87 101 SP-64Ac-QSQQTFSSLWRSLAibQN-NH2 2011.00 1006.53 1006.83 102 SP-65Ac-QSQQTFSNLWSLLA$N-NH2 1940.02 971.01 971.29 103 SP-66Ac-S/SQQ$/FSNLWRLLAibQN-NH2 2037.12 1019.56 1019.78 104 SP-67Ac-QSyQTFVNLWRLLAibQN-NH2 2051.13 1026.57 1026.90 105 SP 68Ac-QSQQ$/FSN$/WRLLAihQN -NH2 2052.09 1027.05 1027.36 106 SP-69Ac-QSQQTFS/NLWS/LLAibQN-NH2 2023.09 1012.55 1013.82 107 SP-70Ac-QSQSTFSSLWRLLAibQN-NH2 1996.09 999.05 999.39 108 SP-71Ac-QSQS/TFSS/LWRLLAibQN-NH2 2024.12 1013.06 1013.37 109 SP-72Ac-QSS/QTFSt//WRLLS/s8QN-NH2 2201.27 1101.64 1102.00 110 SP-73Ac-Sr8SQQTFSSLWRLLAibQN-NH2 2038.14 1020.07 1020.23 111 SP-74Ac-QSQSr8TFSNLWSLLAibQN-NH2 1996.08 999.04 999.32 112 SP-75Ac-QSQQTFSSr8LWRLLASN-NH2 2024.12 1013.06 1013.37 113 SP-76Ac-QS$r5QTFStNLWSLLAibQN-NH2 2032.12 1017.06 1017.39 114 SP-77Ac-S/r8SQQTFS$/LWRLLAibQN-NH2 2066.17 1034.09 1034.80 115 SP-78Ac-QSQS/r8TKSNLWS/LLAibQN-NH2 2024.11 1013.06 1014.34 116 SP-79Ac QSQQMSvYNLWRIIAS/N-NH2 2052.15 1027.08 1027.16 117 SP-80Ac-QSS/r5QTPSt//NLWS/LLAibQN-NH2 2088.18 1045.09 1047.10 118 SP-81Ac-QSQQTFSNLWRLLAihQN-NH2 1988.02 995.01 995.31 119 SP-82HEp/QSQS/TFS/r8NLWRLLS/QN-NH2 2215.29 1108.65 1108.93 120 SP-83Ac ASQQTFSiSNLRWLLSQNNH2 2051.13 1026.57 1026.90 121 SP-84Ac-QSQQTFS/r8NLWRLLS/Q-NH2 2022.14 1012.07 1012.66 122 SP-85Ac-QSQQTFSr8NLWRLLSQ-NH2 1994.11 998.06 998.42 123 SP-86Ac-AAARAASr8AAARAASAA-NH2 1515.90 758.95 759.21 124 SP-87Ac LTFEHYWAQLTSA-NH2 1606.78 804.39 804.59 125 SP-8SAc-LTKSr8HYWAQLSSA-NH2 1668.90 835.45 835.67 126 SP-89Ac-ASQQTKSNLWRLLPQN NH2 1943.00 972.50 973.27 127 SP-90Ac-QS$QTFSTNLW$r5LLAibQN-NH2 2032.12 1017.06 1017.30 128 SP 91Ac QSQQTFAiBNLWRLLAibQN-NH2 1986.04 994.02 99 1.19 129 SP-92Ac-QSQQTFNleNLWRLLNIeQN-NH2 2042.11 1022.06 1022.23 130 SP-93Ac-QSQQTFS/r8NLWRLLAibQN-NH2 2082.14 1042.07 1042.23 131 SP-94Ac-QSQQTFS/r8NLWRLLNIcQN-NH2 2110.17 1056.09 1056.29 132 SP-95Ac-QSQQTKAibNLWRLLS/QN-NH2 2040.09 1021.05 1021.25 133 SP-96Ac-QSQQTFNlENLWRLLS/QN-NH2 2068.12 1035.06 1035.31 134 SP-97Ac-QSQQTF%r8NL6cLWRNleL%QN-NH2 2144.13 1073.07 1073.32 135 SP-98Ac-QSQQTF%r8NLMe6clWRLL%QN-NH2 2158.15 1080.08 1080.31 136 SP-101Ac-FNle$YWE$L-NH2 1160.63 — 1161.70 137 SP-102 Ac-F$r8AYWELL$A-NH21344.75 — 1345.90 138 SP-103 Ac-F$r8AYWQLL$A-NH2 1343.76 — 1344.83 139SP-104 Ac-NlePRF$r8NYWELL$QN-NH2 1925.06 963.53 963.69 140 SP-105Ac-NlePRF$r8DYWRLL$QN-NH2 1953.10 977.55 977.68 141 SP-106Ac-NlePRF$r8NYWRLL$Q-NH2 1838.07 920.04 920.18 142 SP-107Ac-NlePRF$r8NYWRLL$-NH2 1710.01 856.01 856.13 143 SP-108Ac-QSQQTF$r8DLWRLL$QN-NH2 2109.14 1055.57 1055.64 144 SP-109Ac-QSQQTF$r8NLWRLL$EN-NH2 2109.14 1055.57 1055.70 145 SP-110Ac-QSQQTF$r8NLWRLL$QD-NH2 2109.14 1055.57 1055.64 146 SP-111Ac-QSQQTF$r8NLWRLL$S-NH2 1953.08 977.54 977.60 147 SP-112Ac-ESQQTF$r8NLWRLL$QN-NH2 2109.14 1055.57 1055.70 148 SP-113Ac-LTF$r8NLWRNleL$Q-NH2 1635.99 819.00 819.10 149 SP-114Ac-LRF$r8NLWRNleL$Q-NH2 1691.04 846.52 846.68 150 SP-115Ac-QSQQTF$r8NWWRNleL$QN-NH2 2181.15 1091.58 1091.64 151 SP-116Ac-QSQQTF$r8NLWRNleL$Q-NH2 1994.11 998.06 998.07 152 SP-117Ac-QTF$r8NLWRNleL$QN-NH2 1765.00 883.50 883.59 153 SP-118Ac-NlePRF$r8NWWRLL$QN-NH2 1975.13 988.57 988.75 154 SP-119Ac-NlePRF$r8NWWRLL$A-NH2 1804.07 903.04 903.08 155 SP-120Ac-TSFAEYWNLLSP-NH2 1467.70 734.85 734.90 156 SP-121Ac-QTF$r8HWWSQL$S-NH2 1651.85 826.93 827.12 157 SP-122 Ac-FM$YWE$L-NH21178.58 — 1179.64 158 SP-123 Ac-QTFEHWWSQLLS-NH2 1601.76 801.88 801.94159 SP-124 Ac-QSQQTF$r8NLAmwRLNle$QN-NH2 2122.17 1062.09 1062.24 160SP-125 Ac-FMAibY6clWEAc3cL-NH2 1130.47 — 1131.53 161 SP-126Ac-FNle$Y6clWE$L-NH2 1194.59 — 1195.64 162 SP-127Ac-F$zr8AY6clWEAc3cL$z-NH2 1277.63 639.82 1278.71 163 SP-128Ac-F$r8AY6clWEAc3cL$A-NH2 1348.66 — 1350.72 164 SP-129Ac-NlePRF$r8NY6clWRLL$QN-NH2 1986.08 994.04 994.64 165 SP-130Ac-AF$r8AAWALA$A-NH2 1223.71 — 1224.71 166 SP-131 Ac-TF$r8AAWRLA$Q-NH21395.80 698.90 399.04 167 SP-132 Pr-TF$r8AAWRLA$Q-NH2 1409.82 705.91706.04 168 SP-133 Ac-QSQQTF%r8NLWRNleL%QN-NH2 2110.17 1056.09 1056.22169 SP-134 Ac-LTF%r8HYWAQL%SA-NH2 1670.92 836.46 836.58 170 SP-135Ac-NlePRF%r8NYWRLL%QN-NH2 1954.13 978.07 978.19 171 SP-136Ac-NlePRF%r8NY6clWRLL%QN-NH2 1988.09 995.05 995.68 172 SP-137Ac-LTF%r8HY6clWAQL%S-NH2 1633.84 817.92 817.93 173 SP-138Ac-QS%QTF%StNLWRLL%s8QN-NH2 2149.24 1075.62 1075.65 174 SP-139Ac-LTF%r8HY6clWRQL%S-NH2 1718.91 860.46 860.54 175 SP-140Ac-QSQQTF%r8NL6clWRLL%QN-NH2 2144.13 1073.07 1073.64 176 SP-141Ac-%r8SQQTFS%LWRLLAibQN-NH2 2040.15 1021.08 1021.13 177 SP-142Ac-LTF%r8HYWAQL%S-NH2 1599.88 800.94 801.09 178 SP-143Ac-TSF%r8QYWNLL%P-NH2 1602.88 802.44 802.58 179 SP-147Ac-LTI-EHYWAQLTS-NH2 1535.74 768.87 769.5 180 SP-152Ac-F$er8AY6clWEAc3cL$e-NH2 1277.63 639.82 1278.71 181 SP-153Ac-AF$r8AAWALA$A-NH2 1277.63 639.82 1277.84 182 SP-154Ac-TF$r8AAWRLA$Q-NH2 1395.80 698.90 699.04 183 SP-155Pr-TF$r8AAWRLA$Q-NH2 1409.82 705.91 706.04 184 SP-156Ac-LTF$er8HYWAQL$eS-NH2 1597.87 799.94 800.44 185 SP-159Ac-CCPGCCBaQSQQTF$r8NLWREL$QN- 2745.30 1373.65 1372.99 NH2 186 SP-160Ac-CCPGCCBaQSQQTA$r8NLWREL$QN- 2669.27 1335.64 1336.09 NH2 187 SP-161Ac-CCPGCCBaNlePRF$r8NYWREL$QN- 2589.26 1295.63 1296.2 NH2 188 SP-162Ac-LTF$/r8HYWAQL$/S-NH2 1625.90 813.95 814.18 189 SP-163Ac-F%r8HY6clWRAc3cL%-NH2 1372.72 687.36 687.59 190 SP-164Ac-QTF%r8HWWSQL%S-NH2 1653.87 827.94 827.94 191 SP-165Ac-LTA$r8HYWRQL$S-NH2 1606.90 804.45 804.66 192 SP-166Ac-Q$r8QQTFSN$WRLLAibQN-NH2 2080.12 1041.06 1041.61 193 SP-167Ac-QSQQ$r8FSNLWR$LAibQN-NH2 2066.11 1034.06 1034.58 194 SP-168Ac-F$r8AYWEAc3cL$A-NH2 1314.70 658.35 1315.88 195 SP-169Ac-F$r8AYWEAc3cL$S-NH2 1330.70 666.35 1331.87 196 SP-170Ac-F$r8AYWEAc3cL$Q-NH2 1371.72 686.86 1372.72 197 SP-171Ac-F$r8AYWEAibL$S-NH2 1332.71 667.36 1334.83 198 SP-172Ac-F$r8AYWEAL$S-NH2 1318.70 660.35 1319.73 199 SP-173Ac-F$r8AYWEQL$S-NH2 1375.72 688.86 1377.53 200 SP-174Ac-F$r8HYWEQL$S-NH2 1441.74 721.87 1443.48 201 SP-175Ac-F$r8HYWAQL$S-NH2 1383.73 692.87 1385.38 202 SP-176Ac-F$r8HYWAAc3cL$S-NH2 1338.71 670.36 1340.82 203 SP-177Ac-F$r8HYWRAc3cL$S-NH2 1423.78 712.89 713.04 204 SP-178Ac-F$r8AYWEAc3cL#A-NH2 1300.69 651.35 1302.78 205 SP-179Ac-NlePTF%r8NYWREL%QN-NH2 1899.08 950.54 950.56 206 SP-180Ac-TF$r8AAWRAL$Q-NH2 1395.80 698.90 699.13 207 SP-181Ac-TSF%r8HYWAQL%S-NH2 1573.83 787.92 787.98 208 SP-184Ac-F%r8AY6clWEAc3cL%A-NH2 1350.68 676.34 676.91 209 SP-185Ac-LTF$r8HYWAQI$S-NH2 1597.87 799.94 800.07 210 SP-186Ac-LTF$r8HYWAQNle$S-NH2 1597.87 799.94 800.07 211 SP-187Ac-LTF$r8HYWAQL$A-NH2 1581.87 791.94 792.45 212 SP-188Ac-LTF$r8HYWAQL$Abu-NH2 1595.89 798.95 799.03 213 SP-189Ac-LTF$r8HYWAbuQL$S-NH2 1611.88 806.94 807.47 214 SP-190Ac-LTF$er8AYWAQL$eS-NH2 1531.84 766.92 766.96 215 SP-191Ac-LAF$r8HYWAQL$S-NH2 1567.86 784.93 785.49 216 SP-192Ac-LAF$r8AYWAQL$S-NH2 1501.83 751.92 752.01 217 SP-193Ac-LTF$er8AYWAQL$eA-NH2 1515.85 758.93 758.97 218 SP-194Ac-LAF$r8AYWAQL$A-NH2 1485.84 743.92 744.05 219 SP-195Ac-LTF$r8NLWANleL$Q-NH2 1550.92 776.46 776.61 220 SP-196Ac-LTF$r8NLWANleL$A-NH2 1493.90 747.95 1495.6 221 SP-197Ac-LTF$r8ALWANleL$Q-NH2 1507.92 754.96 755 222 SP-198Ac-LAF$r8NLWANleL$Q-NH2 1520.91 761.46 761.96 223 SP-199Ac-LAF$r8ALWANleL$A-NH2 1420.89 711.45 1421.74 224 SP-200Ac-A$r8AYWEAc3cL$A-NH2 1238.67 620.34 1239.65 225 SP-201Ac-F$r8AYWEAc3cL$AA-NH2 1385.74 693.87 1386.64 226 SP-202Ac-F$r8AYWEAc3cL$Abu-NH2 1328.72 665.36 1330.17 227 SP-203Ac-F$r8AYWEAc3cL$Nle-NH2 1356.75 679.38 1358.22 228 SP-204Ac-F$r5AYWEAc3cL$s8A-NH2 1314.70 658.35 1315.51 229 SP-205Ac-F$AYWEAc3cL$r8A-NH2 1314.70 658.35 1315.66 230 SP-206Ac-F$r8AYWEAc3cI$A-NH2 1314.70 658.35 1316.18 231 SP-207Ac-F$r8AYWEAc3cNle$A-NH2 1314.70 658.35 1315.66 232 SP-208Ac-F$r8AYWEAmlL$A-NH2 1358.76 680.38 1360.21 233 SP-209Ac-F$r8AYWENleL$A-NH2 1344.75 673.38 1345.71 234 SP-210Ac-F$r8AYWQAc3cL$A-NH2 1313.72 657.86 1314.7 235 SP-211Ac-F$r8AYWAAc3cL$A-NH2 1256.70 629.35 1257.56 236 SP-212Ac-F$r8AYWAbuAc3cL$A-NH2 1270.71 636.36 1272.14 237 SP-213Ac-F$r8AYWNleAc3cL$A-NH2 1298.74 650.37 1299.67 238 SP-214Ac-F$r8AbuYWEAc3cL$A-NH2 1328.72 665.36 1329.65 239 SP-215Ac-F$r8NleYWEAc3cL$A-NH2 1356.75 679.38 1358.66 240 SP-2165-FAM-BaLTFEHYWAQLTS-NH2 1922.82 962.41 962.87 241 SP-2175-FAM-BaLTF%r8HYWAQL%S-NH2 1986.96 994.48 994.97 242 SP-218Ac-LTF$r8HYWAQhL$S-NH2 1611.88 806.94 807 243 SP-219Ac-LTF$r8HYWAQT1e$S-NH2 1597.87 799.94 799.97 244 SP-220Ac-LTF$r8HYWAQAdm$S-NH2 1675.91 838.96 839.09 245 SP-221Ac-LTF$r8HYWAQhCha$S-NH2 1651.91 826.96 826.98 246 SP-222Ac-LTF$r8HYWAQCha$S-NH2 1637.90 819.95 820.02 247 SP-223Ac-LTF$r8HYWAc6cQL$S-NH2 1651.91 826.96 826.98 248 SP-224Ac-LTF$r8HYWAc5cQL$S-NH2 1637.90 819.95 820.02 249 SP-225Ac-LThF$r8HYWAQL$S-NH2 1611.88 806.94 807 250 SP-226Ac-LTIgl$r8HYWAQL$S-NH2 1625.90 813.95 812.99 251 SP-227Ac-LTF$r8HYWAQChg$S-NH2 1623.88 812.94 812.99 252 SP-228Ac-LTF$r8HYWAQF$S-NH2 1631.85 816.93 816.99 253 SP-229Ac-LTF$r8HYWAQIgl$S-NH2 1659.88 830.94 829.94 254 SP-230Ac-LTF$r8HYWAQCba$S-NH2 1609.87 805.94 805.96 255 SP-231Ac-LTF$r8HYWAQCpg$S-NH2 1609.87 805.94 805.96 256 SP-232Ac-LTF$r8HhYWAQL$S-NH2 1611.88 806.94 807 257 SP-233Ac-F$r8AYWEAc3chL$A-NH2 1328.72 665.36 665.43 258 SP-234Ac-F$r8AYWEAc3cTle$A-NH2 1314.70 658.35 1315.62 259 SP-235Ac-F$r8AYWEAc3cAdm$A-NH2 1392.75 697.38 697.47 260 SP-236Ac-F$r8AYWEAc3chCha$A-NH2 1368.75 685.38 685.34 261 SP-237Ac-F$r8AYWEAc3cCha$A-NH2 1354.73 678.37 678.38 262 SP-238Ac-F$r8AYWEAc6cL$A-NH2 1356.75 679.38 679.42 263 SP-239Ac-F$r8AYWEAc5cL$A-NH2 1342.73 672.37 672.46 264 SP-240Ac-hF$r8AYWEAc3cL$A-NH2 1328.72 665.36 665.43 265 SP-241Ac-Igl$r8AYWEAc3cL$A-NH2 1342.73 672.37 671.5 266 SP-243Ac-F$r8AYWEAc3cF$A-NH2 1348.69 675.35 675.35 267 SP-244Ac-F$r8AYWEAc3cIgl$A-NH2 1376.72 689.36 688.37 268 SP-245Ac-F$r8AYWEAc3cCba$A-NH2 1326.70 664.35 664.47 269 SP-246Ac-F$r8AYWEAc3cCpg$A-NH2 1326.70 664.35 664.39 270 SP-247Ac-F$r8AhYWEAc3cL$A-NH2 1328.72 665.36 665.43 271 SP-248Ac-F$r8AYWEAc3cL$Q-NH2 1371.72 686.86 1372.87 272 SP-249Ac-F$r8AYWEAibL$A-NH2 1316.72 659.36 1318.18 273 SP-250Ac-F$r8AYWEAL$A-NH2 1302.70 652.35 1303.75 274 SP-251Ac-LAF$r8AYWAAL$A-NH2 1428.82 715.41 715.49 275 SP-252Ac-LTF$r8HYWAAc3cL$S-NH2 1552.84 777.42 777.5 276 SP-253Ac-NleTF$r8HYWAQL$S-NH2 1597.87 799.94 800.04 277 SP-254Ac-VTF$r8HYWAQL$S-NH2 1583.85 792.93 793.04 278 SP-255Ac-FTF$r8HYWAQL$S-NH2 1631.85 816.93 817.02 279 SP-256Ac-WTF$r8HYWAQL$S-NH2 1670.86 836.43 836.85 280 SP-257Ac-RTF$r8HYWAQL$S-NH2 1640.88 821.44 821.9 281 SP-258Ac-KTF$r8HYWAQL$S-NH2 1612.88 807.44 807.91 282 SP-259Ac-LNleF$r8HYWAQL$S-NH2 1609.90 805.95 806.43 283 SP-260Ac-LVF$r8HYWAQL$S-NH2 1595.89 798.95 798.93 284 SP-261Ac-LFF$r8HYWAQL$S-NH2 1643.89 822.95 823.38 285 SP-262Ac-LWF$r8HYWAQL$S-NH2 1682.90 842.45 842.55 286 SP-263Ac-LRF$r8HYWAQL$S-NH2 1652.92 827.46 827.52 287 SP-264Ac-LKF$r8HYWAQL$S-NH2 1624.91 813.46 813.51 288 SP-265Ac-LTF$r8NleYWAQL$S-NH2 1573.89 787.95 788.05 289 SP-266Ac-LTF$r8VYWAQL$S-NH2 1559.88 780.94 780.98 290 SP-267Ac-LTF$r8FYWAQL$S-NH2 1607.88 804.94 805.32 291 SP-268Ac-LTF$r8WYWAQL$S-NH2 1646.89 824.45 824.86 292 SP-269Ac-LTF$r8RYWAQL$S-NH2 1616.91 809.46 809.51 293 SP-270Ac-LTF$r8KYWAQL$S-NH2 1588.90 795.45 795.48 294 SP-271Ac-LTF$r8HNleWAQL$S-NH2 1547.89 774.95 774.98 295 SP-272Ac-LTF$r8HVWAQL$S-NH2 1533.87 767.94 767.95 296 SP-273Ac-LTF$r8HFWAQL$S-NH2 1581.87 791.94 792.3 297 SP-274Ac-LTF$r8HWWAQL$S-NH2 1620.88 811.44 811.54 298 SP-275Ac-LTF$r8HRWAQL$S-NH2 1590.90 796.45 796.52 299 SP-276Ac-LTF$r8HKWAQL$S-NH2 1562.90 782.45 782.53 300 SP-277Ac-LTF$r8HYWNleQL$S-NH2 1639.91 820.96 820.98 301 SP-278Ac-LTF$r8HYWVQL$S-NH2 1625.90 813.95 814.03 302 SP-279Ac-LTF$r8HYWFQL$S-NH2 1673.90 837.95 838.03 303 SP-280Ac-LTF$r8HYWWQL$S-NH2 1712.91 857.46 857.5 304 SP-281Ac-LTF$r8HYWKQL$S-NH2 1654.92 828.46 828.49 305 SP-282Ac-LTF$r8HYWANleL$S-NH2 1582.89 792.45 792.52 306 SP-283Ac-LTF$r8HYWAVL$S-NH2 1568.88 785.44 785.49 307 SP-284Ac-LTF$r8HYWAFL$S-NH2 1616.88 809.44 809.47 308 SP-285Ac-LTF$r8HYWAWL$S-NH2 1655.89 828.95 829 309 SP-286Ac-LTF$r8HYWARL$S-NH2 1625.91 813.96 813.98 310 SP-287Ac-LTF$r8HYWAQL$Nle-NH2 1623.92 812.96 813.39 311 SP-288Ac-LTF$r8HYWAQL$V-NH2 1609.90 805.95 805.99 312 SP-289Ac-LTF$r8HYWAQL$F-NH2 1657.90 829.95 830.26 313 SP-290Ac-LTF$r8HYWAQL$W-NH2 1696.91 849.46 849.5 314 SP-291Ac-LTF$r8HYWAQL$R-NH2 1666.94 834.47 834.56 315 SP-292Ac-LTF$r8HYWAQL$K-NH2 1638.93 820.47 820.49 316 SP-293Ac-Q$r8QQTFSN$WRLLAibQN-NH2 2080.12 1041.06 1041.54 317 SP-294Ac-QSQQ$r8FSNLWR$LAibQN-NH2 2066.11 1034.06 1034.58 318 SP-295Ac-LT2Pa1$r8HYWAQL$S-NH2 1598.86 800.43 800.49 319 SP-296Ac-LT3Pa1$r8HYWAQL$S-NH2 1598.86 800.43 800.49 320 SP-297Ac-LT4Pa1$r8HYWAQL$S-NH2 1598.86 800.43 800.49 321 SP-298Ac-LTF2CF3$r8HYWAQL$S-NH2 1665.85 833.93 834.01 322 SP-299Ac-LTF2CN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 323 SP-300Ac-LTF2Me$r8HYWAQL$S-NH2 1611.88 806.94 807 324 SP-301Ac-LTF3CL$r8HYWAQL$S-NH2 1631.83 816.92 816.99 325 SP-302Ac-LTF4CF3$r8HYWAQL$S-NH2 1665.85 833.93 833.94 326 SP-303Ac-LTF4tBu$r8HYWAQL$S-NH2 1653.93 827.97 828.02 327 SP-304Ac-LTF5F$r8HYWAQL$S-NH2 1687.82 844.91 844.96 328 SP-305Ac-LTF$r8HY3BthAAQL$S-NH2 1614.83 808.42 808.48 329 SP-306Ac-LTF2Br$r8HYWAQL$S-NH2 1675.78 838.89 838.97 330 SP-307Ac-LTF4Br$r8HYWAQL$S-NH2 1675.78 838.89 839.86 331 SP-308Ac-LTF2CL$r8HYWAQL$S-NH2 1631.83 816.92 816.99 332 SP-309Ac-LTF4CL$r8HYWAQL$S-NH2 1631.83 816.92 817.36 333 SP-310Ac-LTF3CN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 334 SP-311Ac-LTF4CN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 335 SP-312Ac-LTF34CL2$r8HYWAQL$S-NH2 1665.79 833.90 833.94 336 SP-313Ac-LTF34F2Sr8HYWAQL$S-NH2 1633.85 817.93 817.95 337 SP-314Ac-LTF35F2$r8HYWAQL$S-NH2 1633.85 817.93 817.95 338 SP-315Ac-LTDip$r8HYWAQL$S-NH2 1673.90 837.95 838.01 339 SP-316Ac-LTF2F$r8HYWAQL$S-NH2 1615.86 808.93 809 340 SP-317Ac-LTF3F$r8HYWAQL$S-NH2 1615.86 808.93 809 341 SP-318Ac-LTF4F$r8HYWAQL$S-NH2 1615.86 808.93 809 342 SP-319Ac-LTF4M8HYWAQL$S-NH2 1723.76 862.88 862.94 343 SP-320Ac-LTF3Me$r8HYWAQL$S-NH2 1611.88 806.94 807.07 344 SP-321Ac-LTF4Me$r8HYWAQL$S-NH2 1611.88 806.94 807 345 SP-322Ac-LT1Nal$r8HYWAQL$S-NH2 1647.88 824.94 824.98 346 SP-323Ac-LT2Nal$r8HYWAQL$S-NH2 1647.88 824.94 825.06 347 SP-324Ac-LTF3CF3$r8HYWAQL$S-NH2 1665.85 833.93 834.01 348 SP-325Ac-LTF4NQ2$r8HYWAQL$S-NH2 1642.85 822.43 822.46 349 SP-326Ac-LTF3NQ2$r8HYWAQL$S-NH2 1642.85 822.43 822.46 350 SP-327Ac-LTF$r82ThiYWAQL$S-NH2 1613.83 807.92 807.96 351 SP-328Ac-LTF$r8HBipWAQL$S-NH2 1657.90 829.95 830.01 352 SP-329Ac-LTF$r8HF4tBuWAQL$S-NH2 1637.93 819.97 820.02 353 SP-330Ac-LTF$r8HF4CF3WAQL$S-NH2 1649.86 825.93 826.02 354 SP-331Ac-LTF$r8HF4CLWAQL$S-NH2 1615.83 808.92 809.37 355 SP-332Ac-LTF$r8HF4MeWAQL$S-NH2 1595.89 798.95 799.01 356 SP-333Ac-LTF$r8HF4BrWAQL$S-NH2 1659.78 830.89 830.98 357 SP-334Ac-LTF$r8HF4CNWAQL$S-NH2 1606.87 804.44 804.56 358 SP-335Ac-LTF$r8HF4NQ2WAQL$S-NH2 1626.86 814.43 814.55 359 SP-336Ac-LTF$r8H1NalWAQL$S-NH2 1631.89 816.95 817.06 360 SP-337Ac-LTF$r8H2NalWAQL$S-NH2 1631.89 816.95 816.99 361 SP-338Ac-LTF$r8HWAQL$S-NH2 1434.80 718.40 718.49 362 SP-339Ac-LTF$r8HY1NalAQL$S-NH2 1608.87 805.44 805.52 363 SP-340Ac-LTF$r8HY2NalAQL$S-NH2 1608.87 805.44 805.52 364 SP-341Ac-LTF$r8HYWAQ1$S-NH2 1597.87 799.94 800.07 365 SP-342Ac-LTF$r8HYWAQNle$S-NH2 1597.87 799.94 800.44 366 SP-343Ac-LTF$er8HYWAQL$eA-NH2 1581.87 791.94 791.98 367 SP-344Ac-LTF$r8HYWAQL$Abu-NH2 1595.89 798.95 799.03 368 SP-345Ac-LTF$r8HYWAbuQL$S-NH2 1611.88 806.94 804.47 369 SP-346Ac-LAF$r8HYWAQL$S-NH2 1567.86 784.93 785.49 370 SP-347Ac-LTF$r8NLWANleL$Q-NH2 1550.92 776.46 777.5 371 SP-348Ac-LTF$r8ALWANleL$Q-NH2 1507.92 754.96 755.52 372 SP-349Ac-LAF$r8NLWANleL$Q-NH2 1520.91 761.46 762.48 373 SP-350Ac-F$r8AYWAAc3cL$A-NH2 1256.70 629.35 1257.56 374 SP-351Ac-LTF$r8AYWAAL$S-NH2 1474.82 738.41 738.55 375 SP-352Ac-LVF$r8AYWAQL$S-NH2 1529.87 765.94 766 376 SP-353Ac-LTF$r8AYWAbuQL$S-NH2 1545.86 773.93 773.92 377 SP-354Ac-LTF$r8AYWNleQL$S-NH2 1573.89 787.95 788.17 378 SP-355Ac-LTF$r8AbuYWAQL$S-NH2 1545.86 773.93 773.99 379 SP-356Ac-LTF$r8AYWHQL$S-NH2 1597.87 799.94 799.97 380 SP-357Ac-LTF$r8AYWKQL$S-NH2 1588.90 795.45 795.53 381 SP-358Ac-LTF$r8AYWQQL$S-NH2 1574.89 788.45 788.5 382 SP-359Ac-LTF$r8AYWRQL$S-NH2 1616.91 809.46 809.51 383 SP-360Ac-LTF$r8AYWSQL$S-NH2 1547.84 774.92 774.96 384 SP-361Ac-LTF$r8AYWRAL$S-NH2 1559.89 780.95 780.95 385 SP-362Ac-LTF$r8AYWRQL$A-NH2 1600.91 801.46 801.52 386 SP-363Ac-LTF$r8AYWRAL$A-NH2 1543.89 772.95 773.03 387 SP-364Ac-LTF$r5HYWAQL$s8S-NH2 1597.87 799.94 799.97 388 SP-365Ac-LTF$HYWAQL$r8S-NH2 1597.87 799.94 799.97 389 SP-366Ac-LTF$r8HYWAAL$S-NH2 1540.84 771.42 771.48 390 SP-367Ac-LTF$r8HYWAAbuL$S-NH2 1554.86 778.43 778.51 391 SP-368Ac-LTF$r8HYWALL$S-NH2 1582.89 792.45 792.49 392 SP-369Ac-F$r8AYWHAL$A-NH2 1310.72 656.36 656.4 393 SP-370 Ac-F$r8AYWAAL$A-NH21244.70 623.35 1245.61 394 SP-371 Ac-F$r8AYWSAL$A-NH2 1260.69 631.351261.6 395 SP-372 Ac-F$r8AYWRAL$A-NH2 1329.76 665.88 1330.72 396 SP-373Ac-F$r8AYWKAL$A-NH2 1301.75 651.88 1302.67 397 SP-374Ac-F$r8AYWQAL$A-NH2 1287.74 644.87 1289.13 398 SP-375Ac-F$r8VYWEAc3cL$A-NH2 1342.73 672.37 1343.67 399 SP-376Ac-F$r8FYWEAc3cL$A-NH2 1390.73 696.37 1392.14 400 SP-377Ac-F$r8WYWEAc3cL$A-NH2 1429.74 715.87 1431.44 401 SP-378Ac-F$r8RYWEAc3cL$A-NH2 1399.77 700.89 700.95 402 SP-379Ac-F$r8KYWEAc3cL$A-NH2 1371.76 686.88 686.97 403 SP-380Ac-F$r8ANleWEAc3cL$A-NH2 1264.72 633.36 1265.59 404 SP-381Ac-F$r8AVWEAc3cL$A-NH2 1250.71 626.36 1252.2 405 SP-382Ac-F$r8AFWEAc3cL$A-NH2 1298.71 650.36 1299.64 406 SP-383Ac-F$r8AWWEAc3cL$A-NH2 1337.72 669.86 1338.64 407 SP-384Ac-F$r8ARWEAc3cL$A-NH2 1307.74 654.87 655 408 SP-385Ac-F$r8AKWEAc3cL$A-NH2 1279.73 640.87 641.01 409 SP-386Ac-F$r8AYWVAc3cL$A-NH2 1284.73 643.37 643.38 410 SP-387Ac-F$r8AYWFAc3cL$A-NH2 1332.73 667.37 667.43 411 SP-388Ac-F$r8AYWWAc3cL$A-NH2 1371.74 686.87 686.97 412 SP-389Ac-F$r8AYWRAc3cL$A-NH2 1341.76 671.88 671.94 413 SP-390Ac-F$r8AYWKAc3cL$A-NH2 1313.75 657.88 657.88 414 SP-391Ac-F$r8AYWEVL$A-NH2 1330.73 666.37 666.47 415 SP-392 Ac-F$r8AYWEFL$A-NH21378.73 690.37 690.44 416 SP-393 Ac-F$r8AYWEWL$A-NH2 1417.74 709.87709.91 417 SP-394 Ac-F$r8AYWERL$A-NH2 1387.77 694.89 1388.66 418 SP-395Ac-F$r8AYWEKL$A-NH2 1359.76 680.88 1361.21 419 SP-396Ac-F$r8AYWEAc3cL$V-NH2 1342.73 672.37 1343.59 420 SP-397Ac-F$r8AYWEAc3cL$F-NH2 1390.73 696.37 1392.58 421 SP-398Ac-F$r8AYWEAc3cL$W-NH2 1429.74 715.87 1431.29 422 SP-399Ac-F$r8AYWEAc3cL$R-NH2 1399.77 700.89 700.95 423 SP-400Ac-F$r8AYWEAc3cL$K-NH2 1371.76 686.88 686.97 424 SP-401Ac-F$r8AYWEAc3cL$AV-NH2 1413.77 707.89 707.91 425 SP-402Ac-F$r8AYWEAc3cL$AF-NH2 1461.77 731.89 731.96 426 SP-403Ac-F$r8AYWEAc3cL$AW-NH2 1500.78 751.39 751.5 427 SP-404Ac-F$r8AYWEAc3cL$AR-NH2 1470.80 736.40 736.47 428 SP-405Ac-F$r8AYWEAc3cL$AK-NH2 1442.80 722.40 722.41 429 SP-406Ac-F$r8AYWEAc3cL$AH-NH2 1451.76 726.88 726.93 430 SP-407Ac-LTF2NQ2$r8HYWAQL$S-NH2 1642.85 822.43 822.54 431 SP-408Ac-LTA$r8HYAAQL$S-NH2 1406.79 704.40 704.5 432 SP-409Ac-LTF$r8HYAAQL$S-NH2 1482.82 742.41 742.47 433 SP-410Ac-QSQQTF$r8NLWALL$AN-NH2 1966.07 984.04 984.38 434 SP-411Ac-QAibQQTF$r8NLWALL$AN-NH2 1964.09 983.05 983.42 435 SP-412Ac-QAibQQTF$r8ALWALL$AN-NH2 1921.08 961.54 961.59 436 SP-413Ac-AAAATF$r8AAWAAL$AA-NH2 1608.90 805.45 805.52 437 SP-414Ac-F$r8AAWRAL$Q-NH2 1294.76 648.38 648.48 438 SP-415Ac-TF$r8AAWAAL$Q-NH2 1310.74 656.37 1311.62 439 SP-416Ac-TF$r8AAWRAL$A-NH2 1338.78 670.39 670.46 440 SP-417Ac-VF$r8AAWRAL$Q-NH2 1393.82 697.91 697.99 441 SP-418Ac-AF$r8AAWAAL$A-NH2 1223.71 612.86 1224.67 442 SP-420Ac-TF$r8AAWKAL$Q-NH2 1367.80 684.90 684.97 443 SP-421Ac-TF$r8AAWQAL$Q-NH2 1353.78 677.89 678.01 444 SP-422Ac-TF$r8AAWSAL$Q-NH2 1326.73 664.37 664.47 445 SP-423Ac-LTF$r8AAWRAL$Q-NH2 1508.89 755.45 755.49 446 SP-424Ac-F$r8AYWAQL$A-NH2 1301.72 651.86 651.96 447 SP-425 Ac-F$r8AWWAAL$A-NH21267.71 634.86 634.87 448 SP-426 Ac-F$r8AWWAQL$A-NH2 1324.73 663.37663.43 449 SP-427 Ac-F$r8AYWEAL$-NH2 1231.66 616.83 1232.93 450 SP-428Ac-F$r8AYWAAL$-NH2 1173.66 587.83 1175.09 451 SP-429 Ac-F$r8AYWKAL$-NH21230.72 616.36 616.44 452 SP-430 Ac-F$r8AYWQAL$-NH2 1216.70 609.35609.48 453 SP-431 Ac-F$r8AYWQAL$-NH2 1230.68 616.34 616.44 454 SP-432Ac-F$r8AYWAQL$-NH2 1230.68 616.34 616.37 455 SP-433 Ac-F$r8HYWDQL$S-NH21427.72 714.86 714.86 456 SP-434 Ac-F$r8HFWEQL$S-NH2 1425.74 713.87713.98 457 SP-435 Ac-F$r8AYWHQL$S-NH2 1383.73 692.87 692.96 458 SP-436Ac-F$r8AYWKQL$S-NH2 1374.77 688.39 688.45 459 SP-437 Ac-F$r8AYWQQL$S-NH21360.75 681.38 681.49 460 SP-438 Ac-F$r8HYWSQL$S-NH2 1399.73 700.87700.95 461 SP-439 Ac-F$r8HWWEQL$S-NH2 1464.76 733.38 733.44 462 SP-440Ac-F$r8HWWAQL$S-NH2 1406.75 704.38 704.43 463 SP-441 Ac-F$r8AWWHQL$S-NH21406.75 704.38 704.43 464 SP-442 Ac-F$r8AWWKQL$S-NH2 1397.79 699.90699.92 465 SP-443 Ac-F$r8AWWQQL$S-NH2 1383.77 692.89 692.96 466 SP-444Ac-F$r8HWWSQL$S-NH2 1422.75 712.38 712.42 467 SP-445Ac-LTF$r8NYWANleL$Q-NH2 1600.90 801.45 801.52 468 SP-446Ac-LTF$r8NLWAQL$Q-NH2 1565.90 783.95 784.06 469 SP-447Ac-LTF$r8NYWANleL$A-NH2 1543.88 772.94 773.03 470 SP-448Ac-LTF$r8NLWAQL$A-NH2 1508.88 755.44 755.49 471 SP-449Ac-LTF$r8AYWANleL$Q-NH2 1557.90 779.95 780.06 472 SP-450Ac-LTF$r8ALWAQL$Q-NH2 1522.89 762.45 762.45 473 SP-451Ac-LAF$r8NYWANleL$Q-NH2 1570.89 786.45 786.5 474 SP-452Ac-LAF$r8NLWAQL$Q-NH2 1535.89 768.95 769.03 475 SP-453Ac-LAF$r8AYWANleL$A-NH2 1470.86 736.43 736.47 476 SP-454Ac-LAF$r8ALWAQL$A-NH2 1435.86 718.93 719.01 477 SP-455Ac-LAF$r8AYWAAL$A-NH2 1428.82 715.41 715.41 478 SP-456Ac-F$r8AYWEAc3cL$AAib-NH2 1399.75 700.88 700.95 479 SP-457Ac-F$r8AYWAQL$AA-NH2 1372.75 687.38 687.78 480 SP-458Ac-F$r8AYWAAc3cL$AA-NH2 1327.73 664.87 664.84 481 SP-459Ac-F$r8AYWSAc3cL$AA-NH2 1343.73 672.87 672.9 482 SP-460Ac-F$r8AYWEAc3cL$AS-NH2 1401.73 701.87 701.84 483 SP-461Ac-F$r8AYWEAc3cL$AT-NH2 1415.75 708.88 708.87 484 SP-462Ac-F$r8AYWEAc3cL$AL-NH2 1427.79 714.90 714.94 485 SP-463Ac-F$r8AYWEAc3cL$AQ-NH2 1442.76 722.38 722.41 486 SP-464Ac-F$r8AFWEAc3cL$AA-NH2 1369.74 685.87 685.93 487 SP-465Ac-F$r8AWWEAc3cL$AA-NH2 1408.75 705.38 705.39 488 SP-466Ac-F$r8AYWEAc3cL$SA-NH2 1401.73 701.87 701.99 489 SP-467Ac-F$r8AYWEAL$AA-NH2 1373.74 687.87 687.93 490 SP-468Ac-F$r8AYWENleL$AA-NH2 1415.79 708.90 708.94 491 SP-469Ac-F$r8AYWEAc3cL$AbuA-NH2 1399.75 700.88 700.95 492 SP-470Ac-F$r8AYWEAc3cL$NleA-NH2 1427.79 714.90 714.86 493 SP-471Ac-F$r8AYWEAibL$NleA-NH2 1429.80 715.90 715.97 494 SP-472Ac-F$r8AYWEAL$NleA-NH2 1415.79 708.90 708.94 495 SP-473Ac-F$r8AYWENleL$NleA-NH2 1457.83 729.92 729.96 496 SP-474Ac-F$r8AYWEAibL$Abu-NH2 1330.73 666.37 666.39 497 SP-475Ac-F$r8AYWENleL$Abu-NH2 1358.76 680.38 680.39 498 SP-476Ac-F$r8AYWEAL$Abu-NH2 1316.72 659.36 659.36 499 SP-477Ac-LTF$r8AFWAQL$S-NH2 1515.85 758.93 759.12 500 SP-478Ac-LTF$r8AWWAQL$S-NH2 1554.86 778.43 778.51 501 SP-479Ac-LTF$r8AYWAQI$S-NH2 1531.84 766.92 766.96 502 SP-480Ac-LTF$r8AYWAQNle$S-NH2 1531.84 766.92 766.96 503 SP-481Ac-LTF$r8AYWAQL$SA-NH2 1602.88 802.44 802.48 504 SP-482Ac-LTF$r8AWWAQL$A-NH2 1538.87 770.44 770.89 505 SP-483Ac-LTF$r8AYWAQI$A-NH2 1515.85 758.93 759.42 506 SP-484Ac-LTF$r8AYWAQNle$A-NH2 1515.85 758.93 759.42 507 SP-485Ac-LTF$r8AYWAQL$AA-NH2 1586.89 794.45 794.94 508 SP-486Ac-LTF$r8HWWAQL$S-NH2 1620.88 811.44 811.47 509 SP-487Ac-LTF$r8HRWAQL$S-NH2 1590.90 796.45 796.52 510 SP-488Ac-LTF$r8HKWAQL$S-NH2 1562.90 782.45 782.53 511 SP-489Ac-LTF$r8HYWAQL$W-NH2 1696.91 849.46 849.5 512 SP-491Ac-F$r8AYWAbuAL$A-NH2 1258.71 630.36 630.5 513 SP-492Ac-F$r8AbuYWEAL$A-NH2 1316.72 659.36 659.51 514 SP-493Ac-NlePRF%r8NYWRLL%QN-NH2 1954.13 978.07 978.54 515 SP-494Ac-TSF%r8HYWAQL%S-NH2 1573.83 787.92 787.98 516 SP-495Ac-LTF%r8AYWAQL%S-NH2 1533.86 767.93 768 517 SP-496Ac-HTF$r8HYWAQL$S-NH2 1621.84 811.92 811.96 518 SP-497Ac-LHF$r8HYWAQL$S-NH2 1633.88 817.94 818.02 519 SP-498Ac-LTF$r8HHWAQL$S-NH2 1571.86 786.93 786.94 520 SP-499Ac-LTF$r8HYWHQL$S-NH2 1663.89 832.95 832.38 521 SP-500Ac-LTF$r8HYWAHL$S-NH2 1606.87 804.44 804.48 522 SP-501Ac-LTF$r8HYWAQL$H-NH2 1647.89 824.95 824.98 523 SP-502Ac-LTF$r8HYWAQL$S-NHPr 1639.91 820.96 820.98 524 SP-503Ac-LTF$r8HYWAQL$S-NHsBu 1653.93 827.97 828.02 525 SP-504Ac-LTF$r8HYWAQL$S-NHiBu 1653.93 827.97 828.02 526 SP-505Ac-LTF$r8HYWAQL$S-NHBn 1687.91 844.96 844.44 527 SP-506Ac-LTF$r8HYWAQL$S-NHPe 1700.92 851.46 851.99 528 SP-507Ac-LTF$r8HYWAQL$S-NHChx 1679.94 840.97 841.04 529 SP-508Ac-ETF$r8AYWAQL$S-NH2 1547.80 774.90 774.96 530 SP-509Ac-STF$r8AYWAQL$S-NH2 1505.79 753.90 753.94 531 SP-510Ac-TFF$r8AYWAQL$S-NH2 1559.84 780.92 781.25 532 SP-511Ac-LSF$r8AYWAQL$S-NH2 1517.83 759.92 759.93 533 SP-512Ac-LTF$r8EYWAQL$S-NH2 1589.85 795.93 795.97 534 SP-513Ac-LTF$r8SYWAQL$S-NH2 1547.84 774.92 774.96 535 SP-514Ac-LTF$r8AYWEQL$S-NH2 1589.85 795.93 795.9 536 SP-515Ac-LTF$r8AYWAEL$S-NH2 1532.83 767.42 766.96 537 SP-516Ac-LTF$r8AYWASL$S-NH2 1490.82 746.41 746.46 538 SP-517Ac-LTF$r8AYWAQL$E-NH2 1573.85 787.93 787.98 539 SP-518Ac-LTF2CN$r8HYWAQL$S-NH2 1622.86 812.43 812.47 540 SP-519Ac-LTF3CL$r8HYWAQL$S-NH2 1631.83 816.92 816.99 541 SP-520Ac-LTDip$r8HYWAQL$S-NH2 1673.90 837.95 838.01 542 SP-521Ac-LTF$r8HYWAQTle$S-NH2 1597.87 799.94 800.04 543 SP-522Ac-F$r8AY6clWEAL$A-NH2 1336.66 669.33 1338.56 544 SP-523Ac-F$r8AYd16brWEAL$A-NH2 1380.61 691.31 692.2 545 SP-524Ac-F$r8AYd16fWEAL$A-NH2 1320.69 661.35 1321.61 546 SP-525Ac-F$r8AYd14mWEAL$A-NH2 1316.72 659.36 659.36 547 SP-526Ac-F$r8AYd15clWEAL$A-NH2 1336.66 669.33 669.35 548 SP-527Ac-F$r8AYd17mWEAL$A-NH2 1316.72 659.36 659.36 549 SP-528Ac-LTF%r8HYWAQL%A-NH2 1583.89 792.95 793.01 550 SP-529Ac-LTF$r8HCouWAQL$S-NH2 1679.87 840.94 841.38 551 SP-530Ac-LTFEHCouWAQLTS-NH2 1617.75 809.88 809.96 552 SP-531Ac-LTA$r8HCouWAQL$S-NH2 1603.84 802.92 803.36 553 SP-532Ac-F$r8AYWEAL$AbuA-NH2 1387.75 694.88 694.88 554 SP-533Ac-F$r8AYWEAI$AA-NH2 1373.74 687.87 687.93 555 SP-534Ac-F$r8AYWEANle$AA-NH2 1373.74 687.87 687.93 556 SP-535Ac-F$r8AYWEAmlL$AA-NH2 1429.80 715.90 715.97 557 SP-536Ac-F$r8AYWQAL$AA-NH2 1372.75 687.38 687.48 558 SP-537Ac-F$r8AYWAAL$AA-NH2 1315.73 658.87 658.92 559 SP-538Ac-F$r8AYWAbuAL$AA-NH2 1329.75 665.88 665.95 560 SP-539Ac-F$r8AYWNleAL$AA-NH2 1357.78 679.89 679.94 561 SP-540Ac-F$r8AbuYWEAL$AA-NH2 1387.75 694.88 694.96 562 SP-541Ac-F$r8NleYWEAL$AA-NH2 1415.79 708.90 708.94 563 SP-542Ac-F$r8FYWEAL$AA-NH2 1449.77 725.89 725.97 564 SP-543Ac-LTF$r8HYWAQhL$S-NH2 1611.88 806.94 807 565 SP-544Ac-LTF$r8HYWAQAdm$S-NH2 1675.91 838.96 839.04 566 SP-545Ac-LTF$r8HYWAQIgl$S-NH2 1659.88 830.94 829.94 567 SP-546Ac-F$r8AYWAQL$AA-NH2 1372.75 687.38 687.48 568 SP-547Ac-LTF$r8ALWAQL$Q-NH2 1522.89 762.45 762.52 569 SP-548Ac-F$r8AYWEAL$AA-NH2 1373.74 687.87 687.93 570 SP-549Ac-F$r8AYWENleL$AA-NH2 1415.79 708.90 708.94 571 SP-550Ac-F$r8AYWEAibL$Abu-NH2 1330.73 666.37 666.39 572 SP-551Ac-F$r8AYWENleL$Abu-NH2 1358.76 680.38 680.38 573 SP-552Ac-F$r8AYWEAL$Abu-NH2 1316.72 659.36 659.36 574 SP-553Ac-F$r8AYWEAc3cL$AbuA-NH2 1399.75 700.88 700.95 575 SP-554Ac-F$r8AYWEAc3cL$NleA-NH2 1427.79 714.90 715.01 576 SP-555H-LTF$r8AYWAQL$S-NH2 1489.83 745.92 745.95 577 SP-556mdPEG3-LTF$r8AYWAQL$S-NH2 1679.92 840.96 840.97 578 SP-557mdPEG7-LTF$r8AYWAQL$S-NH2 1856.02 929.01 929.03 579 SP-558Ac-F$r8ApmpEt6clWEAL$A-NH2 1470.71 736.36 788.17 580 SP-559Ac-LTF3CL$r8AYWAQL$S-NH2 1565.81 783.91 809.18 581 SP-560Ac-LTF3CL$r8HYWAQL$A-NH2 1615.83 808.92 875.24 582 SP-561Ac-LTF3CL$r8HYWWQL$S-NH2 1746.87 874.44 841.65 583 SP-562Ac-LTF3CL$r8AYWWQL$S-NH2 1680.85 841.43 824.63 584 SP-563Ac-LTF$r8AYWWQL$S-NH2 1646.89 824.45 849.98 585 SP-564Ac-LTF$r8HYWWQL$A-NH2 1696.91 849.46 816.67 586 SP-565Ac-LTF$r8AYWWQL$A-NH2 1630.89 816.45 776.15 587 SP-566Ac-LTF4F$r8AYWAQL$S-NH2 1549.83 775.92 776.15 588 SP-567Ac-LTF2F$r8AYWAQL$S-NH2 1549.83 775.92 776.15 589 SP-568Ac-LTF3F$r8AYWAQL$S-NH2 1549.83 775.92 785.12 590 SP-569Ac-LTF34F2$r8AYWAQL$S-NH2 1567.83 784.92 785.12 591 SP-570Ac-LTF35F2Sr8AYWAQL$S-NH2 1567.83 784.92 1338.74 592 SP-571Ac-F3CL$r8AYWEAL$A-NH2 1336.66 669.33 705.28 593 SP-572Ac-F3CL$r8AYWEAL$AA-NH2 1407.70 704.85 680.11 594 SP-573Ac-F$r8AY6clWEAL$AA-NH2 1407.70 704.85 736.83 595 SP-574Ac-F$r8AY6clWEAL$-NH2 1265.63 633.82 784.1 596 SP-575Ac-LTF$r8HYWAQLSt/S-NH2 16.03 9.02 826.98 597 SP-576Ac-LTF$r8HYWAQL$S-NHsBu 1653.93 827.97 828.02 598 SP-577Ac-STF$r8AYWAQL$S-NH2 1505.79 753.90 753.94 599 SP-578Ac-LTF$r8AYWAEL$S-NH2 1532.83 767.42 767.41 600 SP-579Ac-LTF$r8AYWAQL$E-NH2 1573.85 787.93 787.98 601 SP-580mdPEG3-LTF$r8AYWAQL$S-NH2 1679.92 840.96 840.97 602 SP-581Ac-LTF$r8AYWAQhL$S-NH2 1545.86 773.93 774.31 603 SP-583Ac-LTF$r8AYWAQCha$S-NH2 1571.88 786.94 787.3 604 SP-584Ac-LTF$r8AYWAQChg$S-NH2 1557.86 779.93 780.4 605 SP-585Ac-LTF$r8AYWAQCba$S-NH2 1543.84 772.92 780.13 606 SP-586Ac-LTF$r8AYWAQF$S-NH2 1565.83 783.92 784.2 607 SP-587Ac-LTF4F$r8HYWAQhL$S-NH2 1629.87 815.94 815.36 608 SP-588Ac-LTF4F$r8HYWAQCha$S-NH2 1655.89 828.95 828.39 609 SP-589Ac-LTF4F$r8HYWAQChg$S-NH2 1641.87 821.94 821.35 610 SP-590Ac-LTF4F$r8HYWAQCba$S-NH2 1627.86 814.93 814.32 611 SP-591Ac-LTF4F$r8AYWAQhL$S-NH2 1563.85 782.93 782.36 612 SP-592Ac-LTF4F$r8AYWAQCha$S-NH2 1589.87 795.94 795.38 613 SP-593Ac-LTF4F$r8AYWAQChg$S-NH2 1575.85 788.93 788.35 614 SP-594Ac-LTF4F$r8AYWAQCba$S-NH2 1561.83 781.92 781.39 615 SP-595Ac-LTF3CL$r8AYWAQhL$S-NH2 1579.82 790.91 790.35 616 SP-596Ac-LTF3CL$r8AYWAQCha$S-NH2 1605.84 803.92 803.67 617 SP-597Ac-LTF3CL$r8AYWAQChg$S-NH2 1591.82 796.91 796.34 618 SP-598Ac-LTF3CL$r8AYWAQCba$S-NH2 1577.81 789.91 789.39 619 SP-599Ac-LTF$r8AYWAQhF$S-NH2 1579.84 790.92 791.14 620 SP-600Ac-LTF$r8AYWAQF3CF3$S-NH2 1633.82 817.91 818.15 621 SP-601Ac-LTF$r8AYWAQF3Me$S-NH2 1581.86 791.93 791.32 622 SP-602Ac-LTF$r8AYWAQ1Nal$S-NH2 1615.84 808.92 809.18 623 SP-603Ac-LTF$r8AYWAQBip$S-NH2 1641.86 821.93 822.13 624 SP-604Ac-LTF$r8FYWAQL$A-NH2 1591.88 796.94 797.33 625 SP-605Ac-LTF$r8HYWAQL$S-NHAm 1667.94 834.97 835.92 626 SP-606Ac-LTF$r8HYWAQL$S-NHiAm 1667.94 834.97 835.55 627 SP-607Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1715.94 858.97 859.79 628 SP-608Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1681.96 841.98 842.49 629 SP-610Ac-LTF$r8HYWAQL$S-NHnPr 1639.91 820.96 821.58 630 SP-611Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1707.98 854.99 855.35 631 SP-612Ac-LTF$r8HYWAQL$S-NHHex 1681.96 841.98 842.4 632 SP-613Ac-LTF$r8AYWAQL$S-NHmdPeg2 1633.91 817.96 818.35 633 SP-614Ac-LTF$r8AYWAQL$A-NHmdPeg2 1617.92 809.96 810.3 634 SP-615Ac-LTF$r8AYWAQL$A-NHmdPeg4 1705.97 853.99 854.33 635 SP-616Ac-F$r8AYd14mWEAL$A-NH2 1316.72 659.36 659.44 636 SP-617Ac-F$r8AYd15clWEAL$A-NH2 1336.66 669.33 669.43 637 SP-618Ac-LThF$r8AYWAQL$S-NH2 1545.86 773.93 774.11 638 SP-619Ac-LT2Nal$r8AYWAQL$S-NH2 1581.86 791.93 792.43 639 SP-620Ac-LTA$r8AYWAQL$S-NH2 1455.81 728.91 729.15 640 SP-621Ac-LTF$r8AYWVQL$S-NH2 1559.88 780.94 781.24 641 SP-622Ac-LTF$r8HYWAAL$A-NH2 1524.85 763.43 763.86 642 SP-623Ac-LTF$r8VYWAQL$A-NH2 1543.88 772.94 773.37 643 SP-624Ac-LTF$r8IYWAQL$S-NH2 1573.89 787.95 788.17 644 SP-625Ac-FTF$r8VYWSQL$S-NH2 1609.85 805.93 806.22 645 SP-626Ac-ITF$r8FYWAQL$S-NH2 1607.88 804.94 805.2 646 SP-627Ac-2NalTF$r8VYWSQL$S-NH2 1659.87 830.94 831.2 647 SP-628Ac-ITF$r8LYWSQL$S-NH2 1589.89 795.95 796.13 648 SP-629Ac-FTF$r8FYWAQL$S-NH2 1641.86 821.93 822.13 649 SP-630Ac-WTF$r8VYWAQL$S-NH2 1632.87 817.44 817.69 650 SP-631Ac-WTF$r8WYWAQL$S-NH2 1719.88 860.94 861.36 651 SP-632Ac-VTF$r8AYWSQL$S-NH2 1533.82 767.91 768.19 652 SP-633Ac-WTF$r8FYWSQL$S-NH2 1696.87 849.44 849.7 653 SP-634Ac-FTF$r8IYWAQL$S-NH2 1607.88 804.94 805.2 654 SP-635Ac-WTF$r8VYWSQL$S-NH2 1648.87 825.44 824.8 655 SP-636Ac-FTF$r8LYWSQL$S-NH2 1623.87 812.94 812.8 656 SP-637Ac-YTF$r8FYWSQL$S-NH2 1673.85 837.93 837.8 657 SP-638Ac-LTF$r8AY6clWEAL$A-NH2 1550.79 776.40 776.14 658 SP-639Ac-LTF$r8AY6clWSQL$S-NH2 1581.80 791.90 791.68 659 SP-640Ac-F$r8AY6clWSAL$A-NH2 1294.65 648.33 647.67 660 SP-641Ac-F$r8AY6clWQAL$AA-NH2 1406.72 704.36 703.84 661 SP-642Ac-LHF$r8AYWAQL$S-NH2 1567.86 784.93 785.21 662 SP-643Ac-LTF$r8AYWAQL$S-NH2 1531.84 766.92 767.17 663 SP-644Ac-LTF$r8AHWAQL$S-NH2 1505.84 753.92 754.13 664 SP-645Ac-LTF$r8AYWAHL$S-NH2 1540.84 771.42 771.61 665 SP-646Ac-LTF$r8AYWAQL$H-NH2 1581.87 791.94 792.15 666 SP-647H-LTF$r8AYWAQL$A-NH2 1473.84 737.92 737.29 667 SP-648Ac-HHF$r8AYWAQL$S-NH2 1591.83 796.92 797.35 668 SP-649Ac-aAibWTF$r8VYWSQL$S-NH2 1804.96 903.48 903.64 669 SP-650Ac-AibWTF$r8HYWAQL$S-NH2 1755.91 878.96 879.4 670 SP-651Ac-AibAWTF$r8HYWAQL$S-NH2 1826.95 914.48 914.7 671 SP-652Ac-AVTF$r8HYWAQL$S-NH2 1817.93 909.97 910.1 672 SP-653Ac-AibWWTF$r8HYWAQL$S-NH2 1941.99 972.00 972.2 673 SP-654Ac-WTF$r8LYWSQL$S-NH2 1662.88 832.44 832.8 674 SP-655Ac-WTF$r8NleYWSQL$S-NH2 1662.88 832.44 832.6 675 SP-656Ac-LTF$r8AYWSQL$a-NH2 1531.84 766.92 767.2 676 SP-657Ac-LTF$r8EYWARL$A-NH2 1601.90 801.95 802.1 677 SP-658Ac-LTF$r8EYWAHL$A-NH2 1582.86 792.43 792.6 678 SP-659Ac-aTF$r8AYWAQL$S-NH2 1489.80 745.90 746.08 679 SP-660Ac-AibTF$r8AYWAQL$S-NH2 1503.81 752.91 753.11 680 SP-661Ac-Amt11-$r8AYWAQL$S-NH2 1579.84 790.92 791.14 681 SP-662Ac-AmwTF$r8AYWAQL$S-NH2 1618.86 810.43 810.66 682 SP-663Ac-NmLTF$r8AYWAQL$S-NH2 1545.86 773.93 774.11 683 SP-664Ac-LNmTF$r8AYWAQL$S-NH2 1545.86 773.93 774.11 684 SP-665Ac-LSarF$r8AYWAQL$S-NH2 1501.83 751.92 752.18 685 SP-667Ac-LGF$r8AYWAQL$S-NH2 1487.82 744.91 745.15 686 SP-668Ac-LTNmF$r8AYWAQL$S-NH2 1545.86 773.93 774.2 687 SP-669Ac-TF$r8AYWAQL$S-NH2 1418.76 710.38 710.64 688 SP-670Ac-ETF$r8AYWAQL$A-NH2 1531.81 766.91 767.2 689 SP-671Ac-LTF$r8EYWAQL$A-NH2 1573.85 787.93 788.1 690 SP-672Ac-LT2Nal$r8AYWSQL$S-NH2 1597.85 799.93 800.4 691 SP-673Ac-LTF$r8AYWAAL$S-NH2 1474.82 738.41 738.68 692 SP-674Ac-LTF$r8AYWAQhCha$S-NH2 1585.89 793.95 794.19 693 SP-675Ac-LTF$r8AYWAQChg$S-NH2 1557.86 779.93 780.97 694 SP-676Ac-LTF$r8AYWAQCba$S-NH2 1543.84 772.92 773.19 695 SP-677Ac-LTF$r8AYWAQF3CF3$S-NH2 1633.82 817.91 818.15 696 SP-678Ac-LTF$r8AYWAQ1Nal$S-NH2 1615.84 808.92 809.18 697 SP-679Ac-LTF$r8AYWAQBip$S-NH2 1641.86 821.93 822.32 698 SP-680Ac-LT2Nal$r8AYWAQL$S-NH2 1581.86 791.93 792.15 699 SP-681Ac-LTF$r8AYWVQL$S-NH2 1559.88 780.94 781.62 700 SP-682Ac-LTF$r8AWWAQL$S-NH2 1554.86 778.43 778.65 701 SP-683Ac-FTF$r8VYWSQL$S-NH2 1609.85 805.93 806.12 702 SP-684Ac-ITF$r8FYWAQL$S-NH2 1607.88 804.94 805.2 703 SP-685Ac-ITF$r8LYWSQL$S-NH2 1589.89 795.95 796.22 704 SP-686Ac-FTF$r8FYWAQL$S-NH2 1641.86 821.93 822.41 705 SP-687Ac-VTF$r8AYWSQL$S-NH2 1533.82 767.91 768.19 706 SP-688Ac-LTF$r8AHWAQL$S-NH2 1505.84 753.92 754.31 707 SP-689Ac-LTF$r8AYWAQL$H-NH2 1581.87 791.94 791.94 708 SP-690Ac-LTF$r8AYWAHL$S-NH2 1540.84 771.42 771.61 709 SP-691Ac-aAibWTF$r8VYWSQL$S-NH2 1804.96 903.48 903.9 710 SP-692Ac-AibWTF$r8HYWAQL$S-NH2 1755.91 878.96 879.5 711 SP-693Ac-AibAWTF$r8HYWAQL$S-NH2 1826.95 914.48 914.7 712 SP-694Ac-fWTF$r8HYWAQL$S-NH2 1817.93 909.97 910.2 713 SP-695Ac-AibWWTF$r8HYWAQL$S-NH2 1941.99 972.00 972.7 714 SP-696Ac-WTF$r8LYWSQL$S-NH2 1662.88 832.44 832.7 715 SP-697Ac-WTF$r8NleYWSQL$S-NH2 1662.88 832.44 832.7 716 SP-698Ac-LTF$r8AYWSQL$a-NH2 1531.84 766.92 767.2 717 SP-699Ac-LTF$r8EYWARL$A-NH2 1601.90 801.95 802.2 718 SP-700Ac-LTF$r8EYWAHL$A-NH2 1582.86 792.43 792.6 719 SP-701Ac-aTF$r8AYWAQL$S-NH2 1489.80 745.90 746.1 720 SP-702Ac-AibTF$r8AYWAQL$S-NH2 1503.81 752.91 753.2 721 SP-703Ac-AmfTF$r8AYWAQL$S-NH2 1579.84 790.92 791.2 722 SP-704Ac-AmwTF$r8AYWAQL$S-NH2 1618.86 810.43 810.7 723 SP-705Ac-NmLTF$r8AYWAQL$S-NH2 1545.86 773.93 774.1 724 SP-706Ac-LNmTF$r8AYWAQL$S-NH2 1545.86 773.93 774.4 725 SP-707Ac-LSarF$r8AYWAQL$S-NH2 1501.83 751.92 752.1 726 SP-708Ac-TF$r8AYWAQL$S-NH2 1418.76 710.38 710.8 727 SP-709Ac-ETF$r8AYWAQL$A-NH2 1531.81 766.91 767.4 728 SP-710Ac-LTF$r8EYWAQL$A-NH2 1573.85 787.93 788.2 729 SP-711Ac-WTF$r8VYWSQL$S-NH2 1648.87 825.44 825.2 730 SP-713Ac-YTF$r8FYWSQL$S-NH2 1673.85 837.93 837.3 731 SP-714Ac-F$r8AY6clWSAL$A-NH2 1294.65 648.33 647.74 732 SP-715Ac-ETF$r8EYWVQL$S-NH2 1633.84 817.92 817.36 733 SP-716Ac-ETF$r8EHWAQL$A-NH2 1563.81 782.91 782.36 734 SP-717Ac-ITF$r8EYWAQL$S-NH2 1589.85 795.93 795.38 735 SP-718Ac-ITF$r8EHWVQL$A-NH2 1575.88 788.94 788.42 736 SP-719Ac-ITF$r8EHWAQL$S-NH2 1563.85 782.93 782.43 737 SP-720Ac-LTF4F$r8AYWAQCba$S-NH2 1561.83 781.92 781.32 738 SP-721Ac-LTF3CL$r8AYWAQhL$S-NH2 1579.82 790.91 790.64 739 SP-722Ac-LTF3CL$r8AYWAQCha$S-NH2 1605.84 803.92 803.37 740 SP-723Ac-LTF3CL$r8AYWAQChg$S-NH2 1591.82 796.91 796.27 741 SP-724Ac-LTF3CL$r8AYWAQCba$S-NH2 1577.81 789.91 789.83 742 SP-725Ac-LTF$r8AY6clWSQL$S-NH2 1581.80 791.90 791.75 743 SP-726Ac-LTF4F$r8HYWAQhL$S-NH2 1629.87 815.94 815.36 744 SP-727Ac-LTF4F$r8HYWAQCba$S-NH2 1627.86 814.93 814.32 745 SP-728Ac-LTF4F$r8AYWAQhL$S-NH2 1563.85 782.93 782.36 746 SP-729Ac-LTF4F$r8AYWAQChg$S-NH2 1575.85 788.93 788.35 747 SP-730Ac-ETF$r8EYWVAL$S-NH2 1576.82 789.41 788.79 748 SP-731Ac-ETF$r8EHWAAL$A-NH2 1506.79 754.40 754.8 749 SP-732Ac-ITF$r8EYWAAL$S-NH2 1532.83 767.42 767.75 750 SP-733Ac-ITF$r8EHWVAL$A-NH2 1518.86 760.43 760.81 751 SP-734Ac-ITF$r8EHWAAL$S-NH2 1506.82 754.41 754.8 752 SP-735Pam-LTF$r8EYWAQL$S-NH2 1786.07 894.04 894.48 753 SP-736Pam-ETF$r8EYWAQL$S-NH2 1802.03 902.02 902.34 754 SP-737Ac-LTF$r8AYWLQL$S-NH2 1573.89 787.95 787.39 755 SP-738Ac-LTF$r8EYWLQL$S-NH2 1631.90 816.95 817.33 756 SP-739Ac-LTF$r8EHWLQL$S-NH2 1605.89 803.95 804.29 757 SP-740Ac-LTF$r8VYWAQL$S-NH2 1559.88 780.94 781.34 758 SP-741Ac-LTF$r8AYWSQL$S-NH2 1547.84 774.92 775.33 759 SP-742Ac-ETF$r8AYWAQL$S-NH2 1547.80 774.90 775.7 760 SP-743Ac-LTF$r8EYWAQL$S-NH2 1589.85 795.93 796.33 761 SP-744Ac-LTF$r8HYWAQL$S-NHAm 1667.94 834.97 835.37 762 SP-745Ac-LTF$r8HYWAQL$S-NHiAm 1667.94 834.97 835.27 763 SP-746Ac-LTF$r8HYWAQL$S-NHnPr3Ph 1715.94 858.97 859.42 764 SP-747Ac-LTF$r8HYWAQL$S-NHnBu3,3Me 1681.96 841.98 842.67 765 SP-748Ac-LTF$r8HYWAQL$S-NHnBu 1653.93 827.97 828.24 766 SP-749Ac-LTF$r8HYWAQL$S-NHnPr 1639.91 820.96 821.31 767 SP-750Ac-LTF$r8HYWAQL$S-NHnEt2Ch 1707.98 854.99 855.35 768 SP-751Ac-LTF$r8HYWAQL$S-NHHex 1681.96 841.98 842.4 769 SP-752Ac-LTF$r8AYWAQL$S-NHmdPeg2 1633.91 817.96 855.35 770 SP-753Ac-LTF$r8AYWAQL$A-NHmdPeg2 1617.92 809.96 810.58 771 SP-754Ac-LTF$r5AYWAAL$s8S-NH2 1474.82 738.41 738.79 772 SP-755Ac-LTF$r8AYWCQuQL$S-NH2 1705.88 853.94 854.61 773 SP-756Ac-LTF$r8CQuYWAQL$S-NH2 1705.88 853.94 854.7 774 SP-757Ac-CQuTF$r8AYWAQL$S-NH2 1663.83 832.92 833.33 775 SP-758H-LTF$r8AYWAQL$A-NH2 1473.84 737.92 737.29 776 SP-759Ac-HHF$r8AYWAQL$S-NH2 1591.83 796.92 797.72 777 SP-760Ac-LT2Nal$r8AYWSQL$S-NH2 1597.85 799.93 800.68 778 SP-761Ac-LTF$r8HCouWAQL$S-NH2 1679.87 840.94 841.38 779 SP-762Ac-LTF$r8AYWCou2QL$S-NH2 1789.94 895.97 896.51 780 SP-763Ac-LTF$r8Cou2YWAQL$S-NH2 1789.94 895.97 896.5 781 SP-764Ac-CQu2TF$r8AYWAQL$S-NH2 1747.90 874.95 875.42 782 SP-765Ac-LTF$r8ACou2WAQL$S-NH2 1697.92 849.96 850.82 783 SP-766Dmaac-LTF$r8AYWAQL$S-NH2 1574.89 788.45 788.82 784 SP-767Hexac-LTF$r8AYWAQL$S-NH2 1587.91 794.96 795.11 785 SP-768Napac-LTF$r8AYWAQL$S-NH2 1657.89 829.95 830.36 786 SP-769Pam-LTF$r8AYWAQL$S-NH2 1728.06 865.03 865.45 787 SP-770Ac-LT2Nal$r8HYAAQL$S-NH2 1532.84 767.42 767.61 788 SP-771Ac-LT2Nal$/r8HYWAQMS-NH2 1675.91 838.96 839.1 789 SP-772Ac-LT2Nal$r8HYFAQL$S-NH2 1608.87 805.44 805.9 790 SP-773Ac-LT2Nal$r8HWAAQL$S -NH2 1555.86 778.93 779.08 791 SP-774Ac-LT2Nal$r8HYAWQL$S-NH2 1647.88 824.94 825.04 792 SP-775Ac-LT2Nal$r8HYAAQW$S-NH2 1605.83 803.92 804.05 793 SP-776Ac-LTW$r8HYWAQL$S-NH2 1636.88 819.44 819.95 794 SP-777Ac-LT1Nal$r8HYWAQL$S-NH2 1647.88 824.94 825.41

In the sequences shown above and elsewhere, the following abbreviationsare used: “Nle” represents norleucine, “Aib” represents2-aminoisobutyric acid, “Ac” represents acetyl, and “Pr” representspropionyl. Amino acids represented as “$” are alpha-MeS5-pentenyl-alanine olefin amino acids connected by an all-carbon i toi+4 crosslinker comprising one double bond. Amino acids represented as“$r5” are alpha-Me R5-pentenyl-alanine olefin amino acids connected byan all-carbon i to i+4 crosslinker comprising one double bond. Aminoacids represented as “$s8” are alpha-Me S8-octenyl-alanine olefin aminoacids connected by an all-carbon i to i+7 crosslinker comprising onedouble bond. Amino acids represented as “$r8” are alpha-MeR8-octenyl-alanine olefin amino acids connected by an all-carbon i toi+7 crosslinker comprising one double bond. “Ahx” represents anaminocyclohexyl linker. The crosslinkers are linear all-carboncrosslinker comprising eight or eleven carbon atoms between the alphacarbons of each amino acid. Amino acids represented as “$1” are alpha-MeS5-pentenyl-alanine olefin amino acids that are not connected by anycrosslinker Amino acids represented as “$/r5” are alpha-MeR5-pentenyl-alanine olefin amino acids that are not connected by anycrosslinker Amino acids represented as “$/s8” are alpha-MeS8-octenyl-alanine olefin amino acids that are not connected by anycrosslinker Amino acids represented as “$/r8” are alpha-MeR8-octenyl-alanine olefin amino acids that are not connected by anycrosslinker Amino acids represented as “Amw” are alpha-Me tryptophanamino acids. Amino acids represented as “Aml” are alpha-Me leucine aminoacids. Amino acids represented as “2ff” are 2-fluoro-phenylalanine aminoacids. Amino acids represented as “3ff” are 3-fluoro-phenylalanine aminoacids. Amino acids represented as “St” are amino acids comprising twopentenyl-alanine olefin side chains, each of which is crosslinked toanother amino acid as indicated. Amino acids represented as “SW” areamino acids comprising two pentenyl-alanine olefin side chains that arenot crosslinked. Amino acids represented as “% St” are amino acidscomprising two pentenyl-alanine olefin side chains, each of which iscrosslinked to another amino acid as indicated via fully saturatedhydrocarbon crosslinks.

For example, the compounds represented as SP-72, SP-56 and SP-138 havethe following structures (SEQ ID NOS 109, 93 and 173, respectively, inorder of appearance):

For example, additional compounds have the following structures (SEQ IDNOS 83, 177, 303, 163, 225, 273, 366, 217, 214, 387 and 184,respectively, in order of appearance):

Example 3: Competition Binding ELISA (HDM2 & HDMX)

p53-His6 (“His6” disclosed as SEQ ID NO: 796) protein (30 nM/well) iscoated overnight at room temperature in the wells of a 96-well Immulonplates. On the day of the experiment, plates are washed with 1×PBS-Tween20 (0.05%) using an automated ELISA plate washer, blocked with ELISAMicro well Blocking for 30 minutes at room temperature; excess blockingagent is washed off by washing plates with 1×PBS-Tween 20 (0.05%).Peptides are diluted from 10 mM DMSO stocks to 500 working stocks insterile water, further dilutions made in 0.5% DMSO to keep theconcentration of DMSO constant across the samples. The peptides areadded to wells at 2× desired concentrations in 50 μl volumes, followedby addition of diluted GST-HDM2 or GST-HMDX protein (finalconcentration: 10 nM). Samples are incubated at room temperature for 2h, plates are washed with PBS-Tween 20 (0.05%) prior to adding 100 μl ofHRP-conjugated anti-GST antibody [Hypromatrix, INC] diluted to 0.5 μg/mlin HRP-stabilizing buffer. Post 30 min incubation with detectionantibody, plates are washed and incubated with 100 μl per well of TMB-ESubstrate solution up to 30 minutes; reactions are stopped using 1M HCLand absorbance measured at 450 nm on micro plate reader. Data isanalyzed using Graph Pad PRISM software.

Example 4: SJSA-1 Cell Viability Assay

SJSA1 cells are seeded at the density of 5000 cells/100 μl/well in96-well plates a day prior to assay. On the day of study cells arewashed once with Opti-MEM Media and 90 μL of the Opti-MEM Media is addedto cells. Peptides are diluted from 10 mM DMSO stocks to 500 μM workingstocks in sterile water, further dilutions made in 0.5% DMSO to keep theconcentration of DMSO constant across the samples. The finalconcentration range μM will be 50, 25, 12.5, 6.25, 3.1, 1.56, 0.8 and 0μM in 100 μL final volume per well for peptides. Final highest DMSOconcentration is 0.5% and will be used as the negative control. CaymanChemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positivecontrol. Nutlin was diluted using the same dilution scheme as peptides10 μl of 10× desired concentrations is added to the appropriate well toachieve the final desired concentrations. Cells are then incubated withpeptides for 20-24 h at 37° C. in humidified 5% CO2 atmosphere.Post-incubation period, cell viability is measured using Promega CellTiter-Glo reagents according to manufacturer′ instructions.

Example 5: SJSA-1 p21 Up-Regulation Assay

SJSA1 cells are seeded at the density of 0.8 million cells/2 ml/well in6-well plates a day prior to assay. On the day of study cells are washedonce with Opti-MEM Media and 1350 μL of the Opti-MEM Media is added tocells. Peptides are diluted from 10 mM DMSO stocks to 500 μM workingstocks in sterile water, further dilutions made in 0.5% DMSO to keep theconcentration of DMSO constant across the samples. Final highest DMSOconcentration is 0.5% and is used as the negative control. CaymanChemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positivecontrol. Nutlin is diluted using the same dilution scheme as peptides150 μl of 10× desired concentrations is added to the appropriate well toachieve the final desired concentrations. Cells are then incubated withpeptides for 18-20 h at 37° C. in humidified 5% CO2 atmosphere.Post-incubation period, cells are harvested, washed with 1×PBS (withoutCa++/Mg++) and lysed in 1× Cell lysis buffer (Cell Signalingtechnologies 10× buffer diluted to 1× and supplemented with proteaseinhibitors and Phosphatase inhibitors) on ice for 30 min. Lysates arecentrifuged at 13000 rpm speed in a microfuge at 40 C for 8 min; clearsupernatants are collected and stored at −80° C. till further use. Totalprotein content of the lysates is measured using BCA protein detectionkit and BSA standards from Thermofisher. 25 μg of the total protein isused for p21 detection ELISA assay. Each condition is set in triplicatefor ELISA plate. The ELISA assay protocol is followed as per themanufacturer's instructions. 25 μg total protein used for each well, andeach well is set up in triplicate. Data is analyzed using Graph PadPRISM software.

Example 6: p53 GRIP Assay

Thermo Scientific*BioImage p53-Hdm2 Redistribution Assay monitors theprotein interaction with Hdm2 and cellular translocation of GFP-taggedp53 in response to drug compounds or other stimuli. Recombinant CHO-hIRcells stably express human p53(1-312) fused to the C-terminus ofenhanced green fluorescent protein (EGFP) and PDE4A4-Hdm2(1-124), afusion protein between PDE4A4 and Hdm2(1-124). They provide aready-to-use assay system for measuring the effects of experimentalconditions on the interaction of p53 and Hdm2. Imaging and analysis isperformed with a HCS platform.

CHO-hIR cells are regularly maintained in Ham's F12 media supplementedwith 1% Penicillin-Streptomycin, 0.5 mg/ml Geneticin, 1 mg/ml Zeocin and10% FBS. Cells seeded into 96-well plates at the density of 7000cells/100 μl per well 18-24 hours prior to running the assay usingculture media. The next day, media is refreshed and PD177 is added tocells to the final concentration of 3 μM to activate foci formation.Control wells are kept without PD-177 solution. 24 h post stimulationwith PD177, cells are washed once with Opti-MEM Media and 50 μL of theOpti-MEM Media supplemented with PD-177 (6 μM) is added to cells.Peptides are diluted from 10 mM DMSO stocks to 500 μM working stocks insterile water, further dilutions made in 0.5% DMSO to keep theconcentration of DMSO constant across the samples. Final highest DMSOconcentration is 0.5% and is used as the negative control. CaymanChemicals Cell-Based Assay (−)-Nutlin-3 (10 mM) is used as positivecontrol. Nutlin was diluted using the same dilution scheme as peptides.50 μl of 2× desired concentrations is added to the appropriate well toachieve the final desired concentrations. Cells are then incubated withpeptides for 6 h at 37° C. in humidified 5% CO2 atmosphere.Post-incubation period, cells are fixed by gently aspirating out themedia and adding 150 μl of fixing solution per well for 20 minutes atroom temperature. Fixed cells are washed 4 times with 200 μl PBS perwell each time. At the end of last wash, 100 μl of 1 μM Hoechst stainingsolution is added. Sealed plates incubated for at least 30 min in dark,washed with PBS to remove excess stain and PBS is added to each well.Plates can be stored at 4° C. in dark up to 3 days. The translocation ofp53/HDM2 is imaged using Molecular translocation module on CellomicsArrayscan instrument using 10× objective, XF-100 filter sets for Hoechstand GFP. The output parameters was Mean-CircRINGAveIntenRatio (the ratioof average fluorescence intensities of nucleus and cytoplasm, (wellaverage)). The minimally acceptable number of cells per well used forimage analysis was set to 500 cells.

Example 7: Direct Binding Assay hDM2 with Fluorescence Polarization (FP)

The assay was performed according to the following general protocol:

-   -   1. Dilute hDM2 (In-house, 41 kD) into FP buffer (High salt        buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 10 μM working        stock solution.    -   2. Add 30 μl of 10 μM of protein stock solution into A1 and B1        well of 96-well black HE microplate (Molecular Devices).    -   3. Fill in 30 μl of FP buffer into column A2 to A12, B2 to B12,        C1 to C12, and D1 to D12.    -   4. 2 or 3 fold series dilution of protein stock from A1, B1 into        A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM        concentration at the last dilution point.    -   5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with        DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10        μM with water (dilution 1:10) and then dilute with FP buffer        from 10 μM to 40 nM (dilution 1:250). This is the working        solution which will be a 10 nM concentration in well (dilution        1:4). Keep the diluted FAM labeled peptide in the dark until        use. 6. Add 10 μl of 10 nM of FAM labeled peptide into each well        and incubate, and read at different time points. Kd with        5-FAM-BaLTFEHYWAQLTS-NH₂ SEQ ID NO: 795) is ˜13.38 nM.

Example 8: Competitive Fluorescence Polarization Assay for hDM2

The assay was performed according to the following general protocol:

-   -   1. Dilute hDM2 (In-house, 41 kD) into FP buffer (High salt        buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 84 nM (2×)        working stock solution.    -   2. Add 20 μl of 84 nM (2×) of protein stock solution into each        well of 96-well black HE microplate (Molecular Devices)    -   3. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with        DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10        μM with water (dilution 1:10) and then dilute with FP buffer        from 10 μM to 40 nM (dilution 1:250). This is the working        solution which will be a 10 nM concentration in well (dilution        1:4). Keep the diluted FAM labeled peptide in the dark until        use.    -   4. Make unlabeled peptide dose plate with FP buffer starting        with 1 μM (final) of peptide and making 5 fold serial dilutions        for 6 points using following dilution scheme.    -   Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution 1:2).        Then, dilute from 5 mM to 500 μM with H₂O (dilution 1:10) and        then dilute with FP buffer from 500 μM to 20 μM (dilution 1:25).        Making 5 fold serial dilutions from 4 μM (4×) for 6 points.    -   5. Transfer 10 μl of serial diluted unlabeled peptides to each        well which is filled with 20 μl of 84 nM of protein.    -   6. Add 10 μl of 10 nM (4×) of FAM labeled peptide into each well        and incubate for 3 hr to read. Results of Examples 7 and 8 are        provided in HDM2 data in FIGS. 7A-D.

Example 9: Direct Binding Assay hDMX with Fluorescence Polarization (FP)

The assay was performed according to the following general protocol:

-   -   1. Dilute hDMX (In-house, 40 kD) into FP buffer (High salt        buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 10 μM working        stock solution.    -   2. Add 30 μl of 10 μM of protein stock solution into A1 and B1        well of 96-well black HE microplate (Molecular Devices).    -   3. Fill in 30 μl of FP buffer into column A2 to A12, B2 to B12,        C1 to C12, and D1 to D12.    -   4. 2 or 3 fold series dilution of protein stock from A1, B1 into        A2, B2; A2, B2 to A3, B3; . . . to reach the single digit nM        concentration at the last dilution point.    -   5. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with        DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10        μM with water (dilution 1:10) and then dilute with FP buffer        from 10 μM to 40 nM (dilution 1:250). This is the working        solution which will be a 10 nM concentration in well (dilution        1:4). Keep the diluted FAM labeled peptide in the dark until        use.    -   6. Add 10 μl of 10 nM of FAM labeled peptide into each well and        incubate, and read at different time points.    -   Kd with 5-FAM-BaLTFEHYWAQLTS-NH₂ (SEQ ID NO: 795) is ˜51 nM.

Example 10: Competitive Fluorescence Polarization Assay for hDMX

The assay was performed according to the following general protocol:

-   -   1. Dilute hDMX (In-house, 40 kD) into FP buffer (High salt        buffer-200 mM Nacl, 5 mM CHAPS, pH 7.5) to make 300 nM (2×)        working stock solution.    -   2. Add 20 μl of 300 nM (2×) of protein stock solution into each        well of 96-well black HE microplate (Molecular Devices)    -   3. Dilute 1 mM (in 100% DMSO) of FAM labeled linear peptide with        DMSO to 100 μM (dilution 1:10). Then, dilute from 100 μM to 10        μM with water (dilution 1:10) and then dilute with FP buffer        from 10 μM to 40 nM (dilution 1:250). This is the working        solution which will be a 10 nM concentration in well (dilution        1:4). Keep the diluted FAM labeled peptide in the dark until        use.    -   4. Make unlabeled peptide dose plate with FP buffer starting        with 5 μM (final) of peptide and making 5 fold serial dilutions        for 6 points using following dilution scheme.    -   5. Dilute 10 mM (in 100% DMSO) with DMSO to 5 mM (dilution 1:2).        Then, dilute from 5 mM to 500 μM with H₂O (dilution 1:10) and        then dilute with FP buffer from 500 μM to 20 μM (dilution 1:25).        Making 5 fold serial dilutions from 20 μM (4×) for 6 points.    -   6. Transfer 10 μl of serial diluted unlabeled peptides to each        well which is filled with 20 μl of 300 nM of protein.    -   7. Add 10 μl of 10 nM (4×) of FAM labeled peptide into each well        and incubate for 3 hr to read.        -   Results of Examples 9 and 10 are provided in HDMX data in            FIGS. 7A-D.

Example 11: Cell Viability Assay

The assay was performed according to the following general protocol:

Cell Plating: Trypsinize, count and seed cells at the pre-determineddensities in 96-well plates a day prior to assay. Following celldensities are used for each cell line in use:

-   -   SJSA-1: 7500 cells/well    -   RKO: 5000 cells/well    -   RKO-E6: 5000 cells/well    -   HCT-116: 5000 cells/well    -   SW-480: 2000 cells/well    -   MCF-7: 5000 cells/well

On the day of study, replace media with fresh media with 11% FBS (assaymedia) at room temperature. Add 180 μL of the assay media per well.Control wells with no cells, receive 200 μl media.

Peptide dilution: all dilutions are made at room temperature and addedto cells at room temperature.

-   -   Prepare 10 mM stocks of the peptides in DMSO. Serially dilute        the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33,        0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the        serially DMSO-diluted peptides 33.3 times using sterile water.        This gives range of 10× working stocks. Also prepare        DMSO/sterile water (3% DMSO) mix for control wells.    -   Thus the working stocks concentration range μM will be 300, 100,        30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using        multichannel.    -   Row H has controls. H1-H3 will receive 20 ul of assay media.        H4-H9 will receive 20 ul of 3% DMSO-water vehicle. H10-H12 will        have media alone control with no cells.    -   Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10        mM) is used as positive control. Nutlin was diluted using the        same dilution scheme as peptides.

Addition of working stocks to cells:

-   -   Add 20 μl of 10× desired concentration to appropriate well to        achieve the final concentrations in total 200 μl volume in well.        (20 μl of 300 μM peptide+180 μl of cells in media=30 μM final        concentration in 200 μl volume in wells). Mix gently a few times        using pipette. Thus final concentration range used will be 30,        10, 3, 1, 0.3, 0.1, 0.03 & 0 μM (for potent peptides further        dilutions are included).    -   Controls include wells that get no peptides but contain the same        concentration of DMSO as the wells containing the peptides, and        wells containing NO CELLS.    -   Incubate for 72 hours at 37° C. in humidified 5% CO₂ atmosphere.    -   The viability of cells is determined using MTT reagent from        Promega. Viability of SJSA-1, RKO, RKO-E6, HCT-116 cells is        determined on day 3, MCF-7 cells on day 5 and SW-480 cells on        day 6. At the end of designated incubation time, allow the        plates to come to room temperature. Remove 80 μl of assay media        from each well. Add 15 μl of thawed MTT reagent to each well.    -   Allow plate to incubate for 2 h at 37° C. in humidified 5% CO₂        atmosphere and add 100 μl solubilization reagent as per        manufacturer's protocol. Incubate with agitation for 1 h at room        temperature and read on Synergy Biotek multiplate reader for        absorbance at 570 nM.    -   Analyze the cell viability against the DMSO controls using        GraphPad PRISM analysis tools.

Reagents:

-   -   Invitrogen cell culture Media        -   i. Falcon 96-well clear cell culture treated plates (Nunc            353072)    -   DMSO (Sigma D 2650)    -   RPMI 1640 (Invitrogen 72400)    -   MTT (Promega G4000)

Instruments:

Multiplate Reader for Absorbance Readout (Synergy 2)

Results of Example 11 are provided in SJSA-1 EC50 data in FIGS. 7A-D.

Example 12: P21 ELISA Assay

The assay was performed according to the following general protocol:

Cell Plating:

-   -   Trypsinize, count and seed SJSA1 cells at the density of 7500        cells/100 μl/well in 96-well plates a day prior to assay.    -   On the day of study, replace media with fresh RPMI-11% FBS        (assay media). Add 90 μL of the assay media per well. Control        wells with no cells, receive 100 μl media.

Peptide Dilution:

-   -   Prepare 10 mM stocks of the peptides in DMSO. Serially dilute        the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33,        0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the        serially DMSO-diluted peptides 33.3 times using sterile water        This gives range of 10× working stocks. Also prepare        DMSO/sterile water (3% DMSO) mix for control wells.    -   Thus the working stocks concentration range μM will be 300, 100,        30, 10, 3, 1, 0.3 and 0 Mix well at each dilution step using        multichannel.    -   Row H has controls. H1-H3 will receive 10 ul of assay media.        H4-H9 will receive 10 ul of 3% DMSO-water vehicle. H10-H12 will        have media alone control with no cells.    -   Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10        mM) is used as positive control. Nutlin was diluted using the        same dilution scheme as peptides.

Addition of Working Stocks to Cells:

-   -   Add 10 μl of 10× desired concentration to appropriate well to        achieve the final concentrations in total 100 μl volume in well.        (10 μl of 300 μM peptide+90 μl of cells in media=30 μM final        concentration in 100 μl volume in wells). Thus final        concentration range used will be 30, 10, 3, 1, 0.3& 0 μM.    -   Controls will include wells that get no peptides but contain the        same concentration of DMSO as the wells containing the peptides,        and wells containing NO CELLS.    -   20 h-post incubation, aspirate the media; wash cells with 1×PBS        (without Ca⁺⁺/Mg⁺⁺) and lyse in 60 μl of 1× Cell lysis buffer        (Cell Signaling technologies 10× buffer diluted to 1× and        supplemented with protease inhibitors and Phosphatase        inhibitors) on ice for 30 min    -   Centrifuge plates in at 5000 rpm speed in at 4° C. for 8 min;        collect clear supernatants and freeze at −80° C. till further        use.

Protein Estimation:

-   -   Total protein content of the lysates is measured using BCA        protein detection kit and BSA standards from Thermofisher.        Typically about 6-7 μg protein is expected per well.    -   Use 50 μl of the lysate per well to set up p21 ELISA.

Human Total p21 ELISA:

The ELISA assay protocol is followed as per the manufacturer'sinstructions. 50 μl lysate is used for each well, and each well is setup in triplicate.

Reagents:

-   -   Cell-Based Assay (−)-Nutlin-3 (10 mM): Cayman Chemicals, catalog        #600034    -   OptiMEM, Invitrogen catalog #51985    -   Cell Signaling Lysis Buffer (10×), Cell signaling technology,        Catalog #9803    -   Protease inhibitor Cocktail tablets (mini), Roche Chemicals,        catalog #04693124001    -   Phosphatase inhibitor Cocktail tablet, Roche Chemicals, catalog        #04906837001    -   Human total p21 ELISA kit, R&D Systems, DYC1047-5    -   STOP Solution (1M HCL), Cell Signaling Technologies, Catalog        #7002

Instruments: Micro centrifuge-Eppendorf 5415D and Multiplate Reader forAbsorbance readout (Synergy 2)

Results of Example 12 are provided in p21 data in FIGS. 7A-D.

Example 13: Caspase 3 Detection Assay

The assay was performed according to the following general protocol:

Cell Plating:

Trypsinize, count and seed SJSA1 cells at the density of 7500 cells/100μl/well in 96-well plates a day prior to assay. On the day of study,replace media with fresh RPMI-11% FBS (assay media). Add 180 μL of theassay media per well. Control wells with no cells, receive 200 μl media.

Peptide Dilution:

-   -   Prepare 10 mM stocks of the peptides in DMSO. Serially dilute        the stock using 1:3 dilution scheme to get 10, 3.3, 1.1, 0.33,        0.11, 0.03, 0.01 mM solutions using DMSO as diluents. Dilute the        serially DMSO-diluted peptides 33.3 times using sterile water        This gives range of 10× working stocks. Also prepare        DMSO/sterile water (3% DMSO) mix for control wells.    -   Thus the working stocks concentration range μM will be 300, 100,        30, 10, 3, 1, 0.3 and 0 μM. Mix well at each dilution step using        multichannel. Add 20 ul of 10× working stocks to appropriate        wells.    -   Row H has controls. H1-H3 will receive 20 ul of assay media.        H4-H9 will receive 20 ul of 3% DMSO-water vehicle. H10-H12 will        have media alone control with no cells.    -   Positive control: HDM2 small molecule inhibitor, Nutlin-3a (10        mM) is used as positive control. Nutlin was diluted using the        same dilution scheme as peptides.

Addition of Working Stocks to Cells:

-   -   Add 10 μl of 10× desired concentration to appropriate well to        achieve the final concentrations in total 100 μl volume in well.        (10 μl of 300 μM peptide+90 μl of cells in media=30 μM final        concentration in 100 μl volume in wells). Thus final        concentration range used will be 30, 10, 3, 1, 0.3& 0 μM.    -   Controls will include wells that get no peptides but contain the        same concentration of DMSO as the wells containing the peptides,        and wells containing NO CELLS.    -   48 h-post incubation, aspirate 80 μl media from each well; add        100 μl Caspase3/7 Glo assay reagent (Promega Caspase 3/7 glo        assay system, G8092) per well, incubate with gentle shaking for        1 h at room temperature.    -   read on Synergy Biotek multiplate reader for luminescence.    -   Data is analyzed as Caspase 3 activation over DMSO-treated        cells.        -   Results of Example 13 are provided in p21 data in FIGS.            7A-D.

What is claimed is:
 1. A peptidomimetic macrocycle comprising an aminoacid sequence which is at least 60% identical to the amino acid sequenceof: SEQ ID NO.
 163. 2. The peptidomimetic macrocycle of claim 1, whereinthe peptidomimetic macrocycle comprises an amino acid sequence which isat least 80% identical to the amino acid sequence of: SEQ ID NO.
 163. 3.The peptidomimetic macrocycle of claim 1, wherein the peptidomimeticmacrocycle comprises an amino acid sequence which is at least 90%identical to the amino acid sequence of: SEQ ID NO.
 163. 4. Thepeptidomimetic macrocycle of claim 1, wherein the peptidomimeticmacrocycle comprises an amino acid sequence which is at least 95%identical to the amino acid sequence of: SEQ ID NO.
 163. 5. Thepeptidomimetic macrocycle of claim 1, wherein the peptidomimeticmacrocycle comprises an amino acid sequence which is: SEQ ID NO.
 163. 6.The peptidomimetic macrocycle of claim 1, wherein the peptidomimeticmacrocycle comprises a helix.
 7. The peptidomimetic macrocycle of claim1, wherein the peptidomimetic macrocycle comprises an α-helix.
 8. Thepeptidomimetic macrocycle of claim 1, wherein the peptidomimeticmacrocycle comprises an α,α-disubstituted amino acid.
 9. Thepeptidomimetic macrocycle of claim 1, wherein the peptidomimeticmacrocycle comprises a crosslinker linking the α-positions of at leasttwo amino acids within the peptidomimetic macrocycle.
 10. Thepeptidomimetic macrocycle of claim 9, wherein at least one of the twoamino acids is an α,α-disubstituted amino acid.
 11. The peptidomimeticmacrocycle of claim 1, wherein the peptidomimetic macrocycle has theformula:

wherein: each A, C, D, and E is independently a natural or non-naturalamino acid, and each D and E independently optionally includes a cappinggroup; each B is independently a natural or non-natural amino acid,amino acid analog, or

each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl,unsubstituted or substituted with halo-; each R₃ is independentlyhydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl,optionally substituted with R₅; each L is independently amacrocycle-forming linker of the formula -L₁-L₂-; each L₁ and L₂ isindependently alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or[—R₄—K—R₄—], each optionally substituted with R₅; each R₄ isindependently alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K isindependently O, S, SO, SO₂, CO, CO₂, or CONR₃; each R₅ is independentlyhalogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, afluorescent moiety, a radioisotope or a therapeutic agent; each R₆ isindependently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeuticagent; each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, each optionally substituted with R₅, or forms partof a cyclic structure with a D residue; each R₈ is independently —H,alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl,cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, eachoptionally substituted with R₅, or forms part of a cyclic structure withan E residue; each v and w is independently an integer from 1-1000; u isan integer from 1-10; each x, y and z is independently an integer from0-10; and each n is independently an integer from 1-5.
 12. Thepeptidomimetic macrocycle of claim 11, wherein L does not include athioether or a triazole.
 13. The peptidomimetic macrocycle of claim 1,wherein the peptidomimetic macrocycle comprises a cross linker linking abackbone amino group of a first amino acid within the peptidomimeticmacrocycle to a second amino acid within the peptidomimetic macrocycle.14. The peptidomimetic macrocycle of claim 13, wherein thepeptidomimetic macrocycle has the formula (IV) or (IVa):

wherein: each A, C, D, and E is independently a natural or non-naturalamino acid, and each D and E independently optionally includes a cappinggroup; each B is independently a natural or non-natural amino acid,amino acid analog, or

each R₁ and R₂ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, eachnon-H group being unsubstituted or substituted with halo-, or part of acyclic structure with an E residue; each R₃ is independently hydrogen,alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, eachnon-H group being optionally substituted with R₅; each L₁ and L₂ isindependently alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or[—R₄—K—R₄—], each optionally substituted with R₅; each R₄ isindependently alkylene, alkenylene, alkynylene, heteroalkylene,cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; each K isindependently O, S, SO, SO₂, CO, CO₂, or CONR₃; each R₅ is independentlyhalogen, alkyl, —OR₆, —N(R₆)₂, —SR₆, —SOR₆, —SO₂R₆, —CO₂R₆, afluorescent moiety, a radioisotope or a therapeutic agent; each R₆ isindependently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeuticagent; each R₇ is independently —H, alkyl, alkenyl, alkynyl, arylalkyl,cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl,or heterocycloaryl, optionally substituted with R₅; each v and w isindependently an integer from 1-1000; u is an integer from 1-10; each x,y and z is independently an integer from 0-10; and each n isindependently an integer from 1-5.
 15. The peptidomimetic macrocycle ofclaim 14, wherein L₁ and L₂ either alone or in combination do notinclude a thioether or a triazole.
 16. The peptidomimetic macrocycle ofclaim 11, wherein L₁ and L₂ are independently alkylene, alkenylene oralkynylene.
 17. The peptidomimetic macrocycle of claim 11, wherein L₁and L₂ are independently C₃-C₁₀ alkylene or C₃-C₁₀ alkenylene.
 18. Thepeptidomimetic macrocycle of claim 17, wherein L₁ and L₂ areindependently C₃-C₆ alkylene or C₃-C₆ alkenylene.
 19. The peptidomimeticmacrocycle of claim 11, wherein R₁ and R₂ are H.
 20. The peptidomimeticmacrocycle of claim 11, wherein R₁ and R₂ are independently alkyl. 21.The peptidomimetic macrocycle of claim 11, wherein R₁ and R₂ are methyl.22. A pharmaceutical composition comprising the peptidomimeticmacrocycle of claim 1.